Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication

ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication Mutation of DNA damage checkpoint signaling kinases ataxia telangiectasia-mutated (ATM) or ATM- and Rad3-related (ATR) results in genomic instability disorders. However, it is not well understood how the instability observed in these syndromes relates to DNA replication/repair defects and failed checkpoint control of cell cycling. As a simple model to address this question, we have studied SV40 chromatin replication in infected cells in the presence of inhibitors of ATM and ATR activities. Two-dimensional gel electrophoresis and southern blotting of SV40 chromatin replication products reveal that ATM activity prevents accumulation of unidirectional replication products, implying that ATM promotes repair of replication-associated double strand breaks. ATR activity alleviates breakage of a functional fork as it converges with a stalled fork. The results suggest that during SV40 chromatin replication, endogenous replication stress activates ATM and ATR signaling, orchestrating the assembly of genome maintenance machinery on viral replication intermediates. Citation: Sowd GA, Li NY, Fanning E (2013) ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication. PLoS Pathog 9(4): e1003283. doi:10.1371/journal.ppat.1003283 Editor: Michael J. Imperiale, University of Michigan, United States of America Received November 27, 2012; Accepted February 14, 2013; Published April 4, 2013 Copyright:  2013 Sowd et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants (GM52948 to EF, T32 AI089554 to JE Crowe, P30 CA068485 to the Vanderbilt-Ingram Cancer Center), and Vanderbilt University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Interestingly, multiple animal viruses have evolved to manipu- Introduction late DNA damage signaling pathways to facilitate viral propaga- Faithful duplication of the genome is vital for cell proliferation. tion [14]. Some viruses, e.g. Herpes simplex, evade or disable In metazoans, the consequences of inaccurate genome replication DNA damage response pathways that result in inappropriate include cell death, premature aging syndromes, neuro-degenera- processing of viral DNA [15,16]. In other cases, viral infection tion disorders, and susceptibility to cancer [1,2]. The DNA appears to activate checkpoint signaling and harness it to promote damage signaling protein kinases ataxia telangiectasia-mutated the infection. HIV, human papillomaviruses, and polyomaviruses (ATM) and ATM- and Rad3-related kinase (ATR), members of induce and depend on ATM signaling for viral propagation the phosphoinositide-3 kinase-like kinase (PIKK) family, act to [17,18,19,20,21,22]. However, mechanistic understanding of how ensure that cells with incompletely replicated or damaged DNA do these viruses activate damage signaling and exploit it for viral not progress through the cell cycle [1]. ATM and DNA-dependent propagation is limited. protein kinase (DNA-PK) respond primarily to DNA double Simian Virus 40 (SV40), a polyomavirus that propagates in strand breaks (DSB) that are associated with either Mre11/NBS1/ monkey kidney cells, has served as a powerful model to study Rad50 (MRN) [3] or Ku70/80 [4], respectively. Additionally, eukaryotic replication proteins and mechanisms in vivo and in vitro intracellular oxidation or alterations in chromatin structure can [23,24,25,26,27]. Checkpoint signaling proteins are dispensable for activate ATM kinase [5,6]. In contrast, single-stranded DNA SV40 DNA replication in vitro, yet in infected cells, ATM or ATR (ssDNA) bound by RPA activate ATR [7,8]. When activated, knockdown, over-expression of kinase-dead variant proteins, or ATM and ATR phosphorylate consensus SQ/TQ motifs in target chemical inhibition of checkpoint signaling clearly decreases or proteins at sites of damage, e.g. the histone H2AX, which delays SV40 chromatin replication [26,28,29,30]. To determine facilitates recruitment of repair proteins and activation of how checkpoint signaling facilitates viral replication in SV40- downstream kinases Chk1 and Chk2 that enforce the checkpoint [8,9]. infected primate cells, we have utilized small molecule inhibitors of the PIKK family members ATM, ATR, and DNA-PK to suppress Failure to activate DNA damage checkpoints results in genome instability syndromes. Mutations in the human ATM gene can checkpoint signaling in host cells during three specific time windows cause the cancer-prone disorder ataxia telangiectasia. Hypomor- after SV40 infection. Characterization of the resulting viral DNA replication products reveals that inhibition of ATM or ATR, but not phic mutations in the ATR gene can cause the genomic instability disorder Seckel Syndrome, but complete loss of ATR results in cell DNA-PK, reduced the yield of unit length viral replication products and caused aberrant viral DNA species to accumulate. ATM death [10,11]. The central roles of ATM and ATR in genome maintenance suggest the potential to manipulate their activity for inhibition led to unidirectional SV40 DNA replication and cancer chemotherapy, fueling the development of potent small concatemeric products, whereas ATR inhibition markedly in- molecules that specifically inhibit ATM and ATR activities in creased broken SV40 DNA replication forks. Our results strongly cellulo [12,13]. suggest that unperturbed viral chromatin replication in infected cells PLOS Pathogens | www.plospathogens.org 1 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity We next asked whether SV40 DNA replication itself might Author Summary induce DNA damage signaling in the absence of viral infection. All cells have evolved pathways to maintain the integrity of Toward this end, the plasmids pMini SV40-wt, and its replication- the genetic information stored in their chromosomes. defective variants lacking Tag helicase activity (D474N) [34], or Endogenous and exogenous agents induce mutations and containing a single base pair insertion that inactivates the viral other damage in DNA, most frequently during DNA origin (In-1) [35], were transfected into BSC40 monkey cells replication. Such DNA damage is under surveillance by a (Figure 1B). As expected, all three plasmids expressed Tag, but complex network of proteins that interact with one only the SV40-wt plasmid replicated (Figure 1C, D). SV40-wt another to signal damage, arrest DNA replication, and activated phosphorylation of Chk1 and Chk2 more robustly than restore genomic integrity before replication resumes. either of the replication-defective constructs (Figure 1C, compare Many viruses that replicate in the nucleus of mammalian lane 1 to lanes 2–3). Moreover, prominent cH2AX foci, a marker host cells have evolved to disable or evade this surveil- of DNA damage signaling in chromatin [36], colocalized with lance system, but others, e.g. polyomaviruses like SV40, chromatin-bound Tag in viral replication centers in SV40-wt activate it and somehow harness it to facilitate robust transfected cells (Figure 1E). In contrast, the few cH2AX foci replication of viral progeny. We have sought to determine detected in cells transfected with the replication defective plasmids how SV40 induces and deploys host DNA damage did not colocalize with Tag. Thus, in the context of transfected signaling in infected cells to promote viral chromosome cells, viral DNA replication, but not SV40-driven Tag expression, replication. Here we present evidence that, like host DNA, is sufficient to induce DNA damage signaling, suggesting that replicating viral DNA suffers damage that activates DNA breaks in replicating viral chromatin may activate check- surveillance and repair pathways. Unlike host replication, point signaling. viral DNA replication persists despite damage signaling, allowing defective replication products to accumulate. In the presence of host DNA damage signaling, these Inhibition of ATM disrupts viral DNA replication centers defective viral products attract proteins of the host To determine the temporal requirements for ATM activity damage surveillance network that correct the defects, during infection, we exposed infected cells to the specific ATM thus maximizing viral propagation. chemical inhibitor Ku-55933 [12] during the early phase (virus entry, Tag expression, host DNA synthesis), late phase (viral DNA results in double strand breaks, activating checkpoint signaling and replication, late gene expression, and virion assembly), or fork repair to generate unit length viral replication products. throughout a 48-hour infection (Figure 2A). Infected cells exposed to the Ku-55933 solvent, DMSO, served as a positive control. Mock-infected cells not treated with inhibitor served as a negative Results control. ATM activity was stimulated by infection, as indicated by SV40 chromatin replication activates DNA damage phosphorylated Nbs1 and Chk2 in western blots (Figure 2B, compare lane 1 to lane 5), reduced by the presence of Ku-55933 in signaling either the early or late phase of infection (Figure 2B, compare Replicating SV40 chromatin in infected cells has been lanes 2, 3 to lane 1), and nearly abolished by the presence of Ku- visualized by fluorescence microscopy in prominent subnuclear 55933 throughout infection (Figure 2B, lane 4). foci that co-localize with Tag and several host proteins essential for To assess the impact of ATM inhibition during each phase of viral DNA replication in vitro, suggesting that these foci may infection on viral chromatin replication, we visualized viral represent viral chromatin replication centers [26,29,31]. However, replication centers and DNA damage signaling in each infected SV40 infection activates ATM and ATR signaling, and several cell population using immunofluorescence microscopy (Figure 2C). DNA damage signaling proteins, e.g. MRN, cH2AX, ATRIP, and In infected cells exposed to DMSO, the normal, brightly stained 53BP1, co-localize with Tag in these foci [28,29,30,32], implying a viral replication centers with colocalized Tag, EdU, and cH2AX link between SV40 replication and damage signaling. On the were observed (Figure 2C). When Ku-55933 was present only other hand, interaction of ectopically expressed Tag with the during the early phase of infection, about half of the cells displayed spindle checkpoint protein Bub1 can also induce cellular normal replication centers with colocalized Tag, EdU and cH2AX chromosome breaks [33], indicating that Tag interference with foci (Figure 2C and D). However, aberrant pan-nuclear staining of host mitotic checkpoint proteins may suffice to damage genomic Tag, EdU, and cH2AX predominated when Ku-55933 was DNA in uninfected cells. present during the late phase or throughout infection (Figure 2C As a first step to assess a potential link between SV40 chromatin and D). Taken together, the results demonstrate that ATM activity replication and DNA damage signaling, viral replication centers in was beneficial but not essential during the early phase of infection, SV40-infected BSC40 monkey cells were characterized in detail. whereas it was vital for the assembly and/or stability of viral Chromatin-bound Tag was visualized in subnuclear foci as replication centers during the late phase of infection. expected and colocalized with newly replicated DNA that had incorporated the deoxynucleoside EdU (Figures 1A and S1A). Inhibition of ATM activity reduces the quantity and Chromatin-bound PCNA, DNA polymerase d, and the clamp- loader RFC, host proteins that are essential for viral DNA quality of viral replication products replication in vitro, colocalized with Tag foci in both BSC40 and The links between ATM activity and SV40 replication centers led human U2OS cells at 48 hours post infection (hpi) (Figures 1A, us to hypothesize that inhibition of ATM might affect not only the S1B–D, and S2). In contrast, Cdc45, an essential component of level, but perhaps also the nature of the viral DNA replication the CMG host replicative helicase that colocalized with replicating products. To investigate this possibility, we used southern blotting to chromatin in mock-infected U2OS cells (Figure S2C, D), was analyze total intracellular DNA from SV40-infected BSC40 cells virtually excluded from viral replication centers (Figures 1A, S1E, that had been treated with DMSO or Ku-55933 throughout and S2C, D). The results strongly suggest that in infected cells, infection (Figure 3A). Inhibition of ATM reduced the level of 5.2 these chromatin-bound Tag foci represent sites of viral, rather kbp viral DNA products migrating as form I (supercoiled), form II than host, chromatin replication. (nicked), and form III (linear), relative to that in the DMSO-treated PLOS Pathogens | www.plospathogens.org 2 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 1. SV40 chromatin replication results in DNA damage signaling. (A) Representative images of chromatin-bound Tag and host DNA replication proteins in SV40- and mock-infected (inset) BSC40 cells at 48 hpi. (B) Features of the SV40 genome and the insertion site of pMini vector [34]. Mutation of Tag residue 474 from D to N abrogates helicase activity [34]. The defective SV40 origin mutant, In-1, features an insertion of a single PLOS Pathogens | www.plospathogens.org 3 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity GC bp in the center of the viral origin allowing Tag binding, but not origin activation [35]. (C, D, E) BSC40 cells transfected with the indicated pMini SV40 plasmids were analyzed by (C) western blot after 24 h, (D) Southern blot of low molecular weight DNA after 48 h [34,73], or (E) immunofluorescence microscopy of chromatin-bound proteins. In (D), SV40 or Mitochondrial probe signal is denoted by SV40 or Mito, respectively. Scale bars in (A) and (E), 10 mm. doi:10.1371/journal.ppat.1003283.g001 control infections (Figure 3A, compare lanes 1–4 to 5–8). However, To quantify the data in Figure 3A, the signal in SV40 monomer ATM inhibition also caused accumulation of high molecular weight bands (forms I, II, and III) in each sample was normalized to that SV40 DNA products too large to enter the gel (Figure 3A, compare of mitochondrial DNA (Mito) in the same sample. This lanes 3, 4 to lanes 7, 8). These large products failed to migrate into normalized monomer signal in each sample was then compared the gel after restriction digestion with enzymes that cut host DNA to that of the normalized monomer bands in the positive control at but not SV40 DNA. In contrast, most of these products collapsed 72 hpi. (Figure 3A, lane 4) and graphed in Figure 3B. The graph into unit length linear SV40 DNA after digestion with an enzyme reveals that ATM inhibition reduced unit length SV40 product by that cleaves SV40 DNA once (Figure S3A), indicating that the large at least 5-fold compared to the DMSO control infections DNA products contain head-to-tail repeats of unit length viral (Figure 3B). Quantification of the concatemeric SV40 DNA in DNA. each sample relative to that of the total SV40 signal in the same Figure 2. ATM inhibition during viral DNA replication disrupts viral replication centers. (A) Experimental scheme for treatment of cells with inhibitor during phases of a 48 h SV40 infection. Early: inhibitor present from 20.5 to 20 hpi. Late: inhibitor present from 20 to 48 hpi. DMSO and Full: solvent or inhibitor, respectively, present from 20.5 to 48 hpi. (B) Western blot of cells treated with Ku-55933 as described in (A). (C) Immunofluorescence of cells treated with Ku-55933 as described in (A) and fixed at 48 hpi. Scale bars, 10 mm. (D) Tag staining patterns, as in (C), were quantified. Graph shows the average of 3 independent experiments. doi:10.1371/journal.ppat.1003283.g002 PLOS Pathogens | www.plospathogens.org 4 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity PLOS Pathogens | www.plospathogens.org 5 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 3. Ku-55933 treatment during viral DNA replication increases aberrant DNA structures. (A) Southern blot of DNA from SV40 infected BSC40 cells in the presence of DMSO or Ku-55933. DMSO or Ku-55933 was present from 30 min prior to infection until cell collection timepoint. M represents Mock-infected cells. (B) Each normalized monomer SV40 form I, II, and III product in (A) was graphed as a fraction of the corresponding normalized monomer produced at 72 hpi in the DMSO control infection. (C) Graph of the percentage of DNA products represented by concatemers in panel (A). (D) Southern blot of SV40 DNA replicated in the presence of Ku-55933 during phases of a 48 h infection in BSC40 cells as explained in Figure 2A. (E) Quantification of total and monomeric SV40 DNA signal normalized to DMSO control from southern blots as in (D). (F, G) Graph of DNA structures (monomer: F and DNA Structure: G) accumulating on southern blots as in (D). Graphs in (E–G) represent 3 to 4 independent experiments. doi:10.1371/journal.ppat.1003283.g003 sample revealed that ATM inhibition increased accumulation of the bubble arc was absent and the unit-mass viral DNA migrated in the 1 n spot as expected (Figure 4A–C). Also as expected, an viral DNA concatemers by an order of magnitude compared to that in the DMSO control samples (Figure 3C). Thus, inhibition of intense double Y arc indicative of converging forks and an X structure signal indicative of hemi-catenates or Holliday junctions ATM throughout infection reduced monomeric and increased were observed (Figure 4C). In addition, the simple Y arc signal concatemeric SV40 DNA products. revealed some unidirectional replicating forks (Figure 4C) that can To determine what stage of SV40 infection required ATM be most easily explained by rolling circle replication. When activity, total intracellular DNA was extracted from infected BamHI-cleaved DNA from DMSO-treated infected cells was BSC40 cells exposed to Ku-55933 during three time windows, as analyzed by 2 d gel electrophoresis, the bubble arc was detected diagrammed in Figure 2A. The purified DNA was separated by and the double Y arc was absent, as expected (Figure 4D). Similar gel electrophoresis and analyzed in southern blots (Figure 3D). to BglI digestion, both an X structure and a weaker simple Y arc Inhibition of ATM either early or throughout infection reproduc- were present (Figure 4D). ibly reduced the level of total viral DNA and monomeric DNA In contrast, the pattern of BglI-digested viral replication products by 50–80% relative to that generated in the DMSO- intermediates generated in the presence of Ku-55933 displayed treated control infection (Figure 3D, E). Similarly, in the late phase a much fainter double Y arc and a more intense simple Y arc of infection, inhibition reduced viral DNA monomers to a level (compare Figure 4E with C). Similarly, X structures and D-loops, comparable to that observed when ATM was inhibited during the or other complex branched intermediates (red star), were more early phase, yet total viral DNA was only insignificantly decreased prominent when ATM was inhibited (compare Figure 4E with C), compared to DMSO-treated cells (Figure 3D, E). SV40 monomers consistent with increased Holliday junction formation between comprised about 80% of the total viral DNA signal in samples replicating rolling circles [38,39]. Likewise, BamHI-cleaved from infected cells exposed to DMSO or Ku-55933 during early replication intermediates from Ku-55933-treated infections dis- phase (Figure 3F). In contrast, monomers comprised only 64% of played a robust simple Y arc and a corresponding decrease in the the total signal in samples treated with Ku-55933 late or bubble arc (Figure 4F). Moreover, the intense X structure and D- throughout infection (Figure 3F). When Ku-55933 was applied loop arcs were retained (Figure 4F). These patterns suggest that either during the late phase or throughout infection, the fraction of inhibition of ATM sharply increased the frequency of rolling circle total viral DNA in concatemers increased 10- and 11-fold, replication (Figure 4G). Quantification of the signal present in the respectively, relative to the fraction in DMSO-treated infected simple Y, X structure, D-loop, and double Y arcs from BglI- cells (Figure 3G). The fraction of total SV40 DNA migrating at 20- digested DNA (Figure 4C, E boxes) showed that ATM inhibition kbp linear also increased in cells treated with Ku-55933 late or increased the abundance of simple Ys, X structures, and D-loop throughout infection, relative to that in DMSO-treated control arcs relative to the double Y arc by six, three, and eight-fold, infections (Figure 3G). respectively, from three to four independent experiments To confirm these findings in a different cell background, the (Figure 4H). Analogously, quantification of BamHI-digested temporal requirements for ATM activity were also determined in DNA (Figure 4D, F boxes) revealed ATM inhibition increased SV40-infected human U2OS cells, with similar results (Figure the quantities of simple Ys, X structures, and D-loop arcs relative S3B–E). Taking the results together, we infer that SV40-infected to the bubble arc (Figure 4I). We conclude that the ATM inhibitor cells require ATM signaling, primarily during the late phase of Ku-55933 increased both rolling circle replication and strand infection, to favor production of unit-length genomes rather than invasion events at the expense of bidirectional SV40 chromatin aberrant products. replication. ATM inhibition increases rolling circle DNA replication Caffeine inhibits SV40 chromatin replication and strand invasion The importance of ATM activity in SV40 chromatin replication To better understand how the aberrant viral replication suggested the possibility that other checkpoint kinases might also products arise, we compared replication intermediates generated contribute to viral infection. To further explore this question, we with and without Ku-55933 during the late phase of infection. The treated SV40-infected BSC40 cells with caffeine, a less selective total DNA was first digested with a restriction nuclease that cleaves inhibitor of both ATM and ATR in vitro and of the S/G2 SV40 once in the viral origin (BglI) or once in the region of checkpoints in vivo [40]. Of note, caffeine is structurally unrelated termination (BamHI). Neutral two-dimensional (2 d) gel electro- to the more potent Ku-55933 and ATR inhibitors [12,13]. As phoresis was then used to separate viral replication intermediates expected, caffeine inhibited phosphorylation of Chk1 and Chk2 from the accumulated non-replicating unit-mass SV40 DNA, when present during the late phase or throughout infection (Figure followed by southern blotting using the whole SV40 genome as the S4A, B) but also hyper-activated DNA-PK (Figure S4B, compare probe [37]. Replicating viral DNA is present in the form of lane 1 with lanes 2–4) [41]. Caffeine reduced the level of total viral circular, converging forks known as Cairns intermediates DNA products in SV40-infected BSC40 cells to less than 1% of (Figure 4B). The digestion of Cairns intermediates with BglI or the control level when caffeine was present throughout infection BamHI results in double Ys or bubbles, respectively (Figure 4A, B). (Figure S5A, B). Exposure to caffeine late or throughout infection In the BglI-cleaved DNA from DMSO-treated control infections, reduced the fraction of total viral DNA signal in monomers (form PLOS Pathogens | www.plospathogens.org 6 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity PLOS Pathogens | www.plospathogens.org 7 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 4. ATM inhibition increases recombination and unidirectional replication. (A) Diagram of neutral 2 d gel electrophoresis arcs generated from digested SV40 DNA. (B) Replicating viral DNA extracted from unperturbed SV40-infected cells consists primarily of circular, late replication intermediates called late Cairns intermediates. Digestion of late Cairns intermediates with BglI yields large double Ys, whereas BamHI digestion yields large bubbles. (C, D, E, F) Southern blot of neutral 2 d gel of BglI-cleaved DNA replicated in the presence of DMSO (C, D) or Ku-55933 (E, F) during the late phase of a 48 h SV40 infection in BSC40 cells. DNA was cleaved within the viral origin of replication with BglI (C, E) or the region of fork convergence with BamHI (D, F). The red star denotes an arc representing strand invasion events (D-loops) or highly branched molecules [38,39]. On the simple Y arc in (F), S denotes a replication stall point near the viral origin of replication. Dashed boxes denote regions of each arc quantified in (H) and (I). (G) Concatemers of SV40 DNA that accumulated when ATM was inhibited can arise by either replication- (top) or recombination- (bottom) dependent rolling circle replication. Digestion of replication-dependent rolling circles with BglI or BamHI results in simple Ys of all sizes. Digestion of recombination-dependent rolling circles creates D-loops of all sizes. (H) Graph of DNA signal present on simple Y, double Y,X structure, or D-loop arc divided by DNA signal in the double Y arc from DNA digested with BglI. (I) Graph of DNA signal from BamHI digested DNA in simple Y, bubble, X structure, or D-loop arc divided by DNA signaling in the bubble arc. Each graph in (H) and (I) represents the average of 3 to 4 independent experiments. doi:10.1371/journal.ppat.1003283.g004 I, II, III) and increased the fraction in concatemers and other S8B–D). Taken together, these results indicate that infected cells aberrant products (Figure S5A, C, D). Similarly, in SV40-infected require ATR activity before, as well as during viral chromatin U2OS cells, caffeine reduced total viral replication products and replication, for normal accumulation of viral genomes. increased the fraction of aberrant products (Figure S5E–H). The results further confirm the role of ATM activity in SV40 Broken and/or stalled forks accumulate in ATR-inhibited chromatin replication in infected cells and suggest that ATR SV40-infected cells and/or DNA-PK activity may stimulate viral replication. The structures of viral replication intermediates generated in the presence and absence of ATR kinase activity were characterized DNA-PK activity is dispensable for SV40 chromatin by using neutral 2 d gel electrophoresis and southern blotting. As replication expected, BglI-digested SV40 replication intermediates from Although SV40 infection did not activate DNA-PK, it was control infections displayed a strong double Y arc indicative of activated in infected cells exposed to Ku-55933 or caffeine, as converging forks, X structures, and a weaker simple Y arc with evidenced by DNA break-dependent auto-phosphorylation of both legs of similar intensity (Figure 6B). In contrast, BglI-digested DNA-PK at S2056 [41] (Figures 2B, S4B). To test for a potential replication intermediates from ATRi-treated cells yielded a novel role of DNA-PK activity in viral chromatin replication, SV40- pattern (Figure 6C). Although the double Y and X structure arcs infected BSC40 cells were exposed to small molecule inhibitors of closely resembled those in the DMSO control, the simple Y arc DNA-PK during the early or late phase, or throughout infection displayed much greater intensity in the leg closer to the 1 n linear and total intracellular DNA was analyzed by southern blotting DNA (Figure 6B and C, zoomed box) than in the other leg closer (Figure S6A–C). When DNA-PK was inhibited with either to 2 n linear DNA. This pattern is not consistent with rolling circle Nu7441 or Nu7026, the levels of viral monomer and aberrant replication, which generates a uniformly intense simple Y arc viral DNA products closely resembled those in SV40-infected (Figure 4) or with two stalled replication forks, of which one BSC40 cells (Figure S6D). Moreover, inhibition of DNA-PK had breaks, creating an asymmetric simple Y [42]. The observed little or no effect on viral replication centers (data not shown). pattern is also inconsistent with one normal replication fork and Thus, it is unlikely that DNA-PK has a major role in viral one slower moving fork, which would converge asymmetrically to chromatin replication in unperturbed infected cells. generate a cone-shaped signal between the X structure arc and the Y arc [43]. However, the novel pattern observed could arise if one ATR inhibition decreases SV40 DNA replication fork stalls prematurely (Figure 6F, I, II), while the other fork The role of ATR kinase activity in infection was directly progresses until it encounters the stalled fork and then breaks, examined by treating SV40-infected BSC40 cells with a specific generating a broken late Cairns intermediate (Figure 6F, III, IV) small molecule inhibitor of ATR, VE-821 (ATRi) [13], during [37]. Close inspection of the intense leg of the Y arc reveals that its three different time windows of infection (Figure S7A). As intensity is uneven, suggesting that it may arise from a series of expected, ATRi caused a third of the cells to lose viability over closely spaced break sites along the Y arc (Figure 6C). If the break 48 h, but SV40-infected and mock-infected cells were equally sites reside 2.5 kb or less from the BglI cleavage site, the intensity sensitive (Figure S7B). SV40 infection activated Chk1, as indicated of signals would be greater in the right leg of the simple Y arc, as by phosphorylation of Ser317 (Figure S7C, compare lane 1 with observed (Figure 6C, box). This interpretation predicts that if lane 5), and ATRi effectively suppressed ATR activation during replication products from the ATRi-treated infection were each time window (Figure S7C, lanes 2–4). digested with BamHI, which cleaves 2.5 kb from the BglI site, Viral DNA replication products from the four cell populations the sites of breakage, and hence greater signal intensity, should and mock-infected cells were analyzed by southern blotting and shift to the left leg of the simple Y arc, closer to the 2 n linear DNA quantified relative to mitochondrial DNA in the same samples. In (Figure 6A, E). Indeed, this shift was observed (compare Figure 6D the presence of ATRi, the level of total viral DNA replication with E), confirming that when the moving replication fork products declined markedly relative to that in DMSO-treated encountered a fork that had stalled in the presence of ATRi, the control infections, amounting to only 10% of the control when moving fork broke (Figures 6F and S9). ATRi was present for the full 48 h (Figure 5B, C). In cells exposed to ATRi during the late phase or throughout infection, the fraction Discussion of viral DNA products in monomers (forms I, II and III) dropped, whereas that in concatemers and other aberrant products rose This study presents several lines of evidence that SV40 (Figure 5B–E and Figure S8A). Analysis of viral replication harnesses host DNA damage signaling for quality control of viral products from SV40-infected U2OS cells exposed to ATRi chromatin replication. We show that viral DNA replication in vivo demonstrated a similar requirement for ATR activity (Figure is sufficient to induce DNA damage signaling at viral replication PLOS Pathogens | www.plospathogens.org 8 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 5. ATR is crucial for SV40 chromatin replication. (A) Scheme for application of ATRi during phases of a 48 h SV40 infection. (B) Southern blot of DNA replicated in BSC40 cells when ATRi was present during phases of a 48 h SV40 infection described in (A). (C) Graph of total viral or SV40 monomer DNA signals normalized to SV40 DNA replicated in the presence of DMSO from southern blots as shown in (B). (D, E) Graph of of monomer (D) or aberrant (E) structure(s) accumulated as a result of ATR inhibition from southern blots as shown in (C). Each bar in (C–E) shows the average from 3 to 4 independent experiments. doi:10.1371/journal.ppat.1003283.g005 centers (Figures 1, S1, S2), suggesting that DNA lesions may arise DNA damage signaling nucleates the assembly of SV40 in unperturbed replicating viral DNA. Importantly, damage replication centers signaling is vital to maintain viral replication centers (Figures 1, SV40 chromatin replication centers resemble over-sized host 2). Furthermore, suppression of ATM and/or ATR signaling DNA damage response foci (for a comparison, see Figure 1 in ref increases the level of aberrant viral replication products at the [29]), where diverse damage signaling and DNA repair proteins expense of unit length viral DNA (Figures 3–5, S3, S5, S8), assemble on chromatin at a DNA lesion and dissociate when implying that viral replication-associated damage in infected cells repair is completed [1,44]. Many of the same signaling and repair requires ATM and ATR signaling to promote repair of viral proteins are found at both viral replication centers and host replication forks. Lastly, our results indicate that the defective damage response foci [18,21,22,28,29,30,32,33] (Sowd, unpub- replication intermediates resulting from inhibition of ATM lished). However, unlike the prominent viral replication centers, (Figure 4) and ATR (Figures 6, S9) are distinctive. Taken together, the punctate host damage response foci encompass megabase our results support a model in which ATM and ATR serve regions of chromatin, raising the question of how SV40 mini- different but complementary roles in orchestrating repair at viral chromosomes give rise to the large subnuclear foci observed in the replication forks (Figure 7). microscope. The size of SV40 replication centers increases with PLOS Pathogens | www.plospathogens.org 9 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 6. ATR inhibition results in fork stalling and breakage of converging forks. (A) Schematic of replication intermediate migration patter on a neutral 2 d gel generated from digested SV40 DNA. (B, C, D, E) Southern blot of neutral 2 d gel electrophoresis of BglI- (B, C) or BamHI-cut (D, E) DNA from SV40-infected BSC40 cells exposed to DMSO (B, D) or ATRi (C, E) during the late phase of SV40 infection as described in Figure 5A. (F) Diagrams of replication intermediates on a simple Y arc produced when ATR was inhibited. BamHI (green) and BglI (orange) sites are denoted by colored lines. I. Replication initiates at the origin and proceeds bidirectionally producing theta replication intermediates. II. Replisomes continue replication until one encounters a replication block (red triangle) causing one stalled fork. III. The stalled replication fork is closest to orange BglI site (viral origin of replication). The functional replisome continues replication and converges with the stalled replication fork. IV. One-sided DSB forms at the replicating fork of late Cairns intermediate shown in (III) as it translocates toward the stall site. V. Simple Y created by digestion of the broken late Cairns intermediate shown in (IV) with BglI or BamHI. VI. Diagram of the predicted outcome of the simple Y shown in panel (V) following neutral 2 d gel electrophoresis and southern blotting. The stall point on the simple Y arc (light green circle) corresponds to the simple Y in panel (V). doi:10.1371/journal.ppat.1003283.g006 the number of incoming viral genomes and with time post- [45]. Moreover, unperturbed viral replication centers display infection in permissive primate cells [29], suggesting that our nascent ssDNA (Sowd, unpublished) and DNA breaks that are ability to detect viral replication centers depends on the ability of likely responsible for activating checkpoint signaling, analogous to each infected cell to generate 10–100 thousand daughter genomes lesions that nucleate host damage response foci. PLOS Pathogens | www.plospathogens.org 10 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 7. Model of ATM and ATR functions in SV40 DNA replication. (I) Tag initiates viral DNA replication at the viral origin of replication (blue) and the two replication forks progress bidirectionally (red arrowheads). For simplicity, proteins are not shown. (II) Viral DNA replicates quickly until the forks converge to form a late Cairns intermediate (III), which slowly completes replication. (IV) Topoisomerase IIa decatenates fully replicated DNA molecules, yielding two form I daughter molecules. (V) When ATM is inhibited, a one-ended double strand break at a replication fork leads to loss of the replication machinery, while the other fork continues to replicate DNA, generating a rolling circle (VI). (VII) ATM kinase activity facilitates the repair of one-ended double strand breaks. (VIII) When ATR is inhibited, a stalled replication fork remains stable until a functional replication fork approaches it, generating a broken replication intermediate (IX). (X) ATR kinase activity facilitates convergence of moving fork with the stalled fork. We suggest that in the presence of ATM and ATR, repair proteins act on the defective intermediates V and IX to reassemble an intermediate with two functional forks. doi:10.1371/journal.ppat.1003283.g007 PLOS Pathogens | www.plospathogens.org 11 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity A major difference between SV40 replication centers and host might be inaccurately joined with broken host chromatin, damage response foci is that checkpoint signaling does not inhibit contributing to viral tumorigenesis [55]. the viral replication machinery, whereas Chk2 phosphorylation of the purified host replicative helicase Cdc45/Mcm2-7/GINS How does ATR signaling orchestrate SV40 replication fork inhibits its helicase activity in vitro [46] and Chk1 inhibits Cdc45 convergence? recruitment to chromatin to initiate replication in vivo [47]. Based SV40 chromatin replication was highly sensitive to inhibition of on these considerations, we suggest that SV40 replication centers ATR throughout a 48 h infection (Figures 5, S8). One serve as hubs where host replication and repair factors efficiently consequence of ATR inhibition was that infected cells continued service many client viral genomes in close proximity. These hubs to cycle throughout infection, rather than arresting in late S phase are nucleated and maintained by the assembly of the ATM and where viral DNA replication would be favored [30]. However, the ATR signaling complexes at sites of viral replication stress, most prominent SV40 replication defect induced by ATRi was the followed by recruitment of downstream repair factors [1]. Of note, tendency of converging replication forks to stall and break all of the host proteins needed for SV40 DNA replication in vitro (Figures 6, 7, S9). Our data imply that after initiating replication [23,24,25] also function in host DNA repair [23,25,48,49]. Thus at the viral origin, one replisome encounters an unknown SV40, though it encodes only a single essential replication protein, replication block at variable positions in the viral genome has evolved a rather remarkable strategy to generate viral (Figure 6F, S9, I and II, red triangle). Since the two sister Tag replication compartments. helicases need not remain coupled after initiation, they can proceed asynchronously as they replicate the viral genome bidirectionally [26,56,57,58,59]. Thus, the functional, unstalled ATM signaling orchestrates reassembly of viral replisome continues replication until it approaches the stalled fork replication forks, reducing unidirectional replication forks (Figure 6F, III). We suggest that without ATR activity, the Recent studies in several laboratories, including ours, estab- unstalled fork cannot converge with the stalled fork and breaks, lished that knockdown or inhibition of ATM in polyomavirus- yielding the pattern observed on the simple Y arc (Figure 6C, E, F, infected cells reduced production of unit length viral genomes IV–VI). Consistent with this interpretation, fork convergence is [21,22,28,29]. Since these studies evaluated only unit length viral well known to represent a slow step during unperturbed SV40 DNA, the aberrant viral replication products generated by DNA replication in infected cells and to occur in a ,1 kbp region unidirectional replication forks were overlooked (Figures 3, 4, around the BamHI site [60,61,62], suggesting that specialized host S3). Interestingly, total intracellular DNA from unperturbed proteins and ATR-dependent modifications may be needed to infected CV1P cells has also been reported to contain head-to- complete replication. tail SV40 DNA repeats of 50 to 100 kbp at very late times after Our observation that ATRi renders SV40 fork convergence infection [45]. These observations indicate that concatemers may prone to DNA breakage is reminiscent of common fragile sites in be a normal product of viral replication, and suggests that the human genome, which suffer gaps and breaks in Seckel inhibition of ATM activity might simply increase the frequency of Syndrome cells that express defective ATR alleles [63]. Thus unidirectional replication, advance its timing, or both. SV40 and other small DNA tumor virus genomes may harbor a Although replication-associated breaks may be a rare event potential fragile site in the region where the two viral replication during unperturbed viral DNA replication, the large number of forks converge. Consistent with this speculation, C-terminal replicating viral genomes would facilitate their detection, partic- truncation of the polyomaviral T antigen encoded in the ‘‘fragile ularly when ATM activity is suppressed. Yet surprisingly, when site’’ could render an integrated viral genome replication-defective undigested total intracellular DNA from an ATM-inhibited and perhaps more tumorigenic [52,64,65,66]. Similarly, the viral infection was analyzed by 2 d gel electrophoresis, bidirectional ‘‘fragile site’’ where replication forks converge would correspond replication was still observed (data not shown) and unit length viral to common viral genome breakpoints in integrated high risk DNA remained the predominant product when ATM was papillomaviral genomes in cervical cancer [67,68,69]. inhibited (Figures 3 and S3). These observations can be most simply explained by a model in which theta-form SV40 replication Materials and Methods intermediates (Figure 7, I–III) break, giving rise to unidirectional forks that amplify the break by generating concatemers and For details not described below, please refer to the online branched concatemers [38,39] (Figure 7, V, VI). Our data suggest Supporting Methods (Protocol S1). that ATM kinase activity is crucial for the repair of one-ended replication-associated DSBs to reassemble bidirectional replication Use of PIKK inhibitors intermediates (Figure 7, VII) [49,50,51]. Ku-55933, kindly provided by Astra-Zeneca, was used as It is interesting to consider a possible role for unidirectional viral described [12,29]. Importantly, Ku-55933 did not inhibit sixty off- replication and its large concatemeric products in the tumorigenic target kinases. It specifically inhibits purified ATM with an IC50 activity of SV40, and more broadly of polyoma- and papilloma- of 12.9 nM, whereas it inhibits the related kinases mTOR and viruses. Concatemeric genomes of Merkel cell carcinoma virus and DNA-PK with IC values of 2500 nM and 9300 nM, respective- HPV are often integrated into human chromosomal DNA in ly, in vitro [12]. Caffeine (Sigma) was dissolved to 24 mM in tumors associated with these viruses [52,53,54]. The integration DMEM and used at a final concentration of 8 mM to inhibit events and the consequences of long-term viral oncogene ATM and ATR [40]. ATRi and Nu7441 were generous gifts from expression are primary risk factors for such cancers. It seems Dr. David Cortez. ATRi dissolved in DMSO at 5 mM was used at likely that in an infected cell under conditions of insufficient ATM a final concentration of 5 mM [13]. ATRi selectively inhibits ATR activity, the level of viral concatemers would rise. With inadequate with a K of 13 nM, whereas at least a 100-fold higher ATM activity, breaks in host chromosomal DNA would also be concentration is required in vitro to inhibit the related kinases less frequently repaired through accurate, homology-dependent ATM (K = 16000 nM), DNA-PK (K = 2200 nM), mTOR i i repair. Thus one can speculate that viral DNA concatemers (K = 1000 nM), and PI3Kgamma (K = 3900 nM) [13]. Nu7441 i i generated under conditions of insufficient DNA damage signaling was dissolved in DMSO to 2 mM and applied to cells at 1 mM PLOS Pathogens | www.plospathogens.org 12 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity [70,71]. Nu7026 (EMD) was dissolved to 5 mM in DMSO and normalized signal present in the infected solvent control to yield used at a final concentration of 10 mM [72]. the DNA signal (% of DMSO). DMEM containing inhibitor or solvent was added to cells The southern blot signals from an equal area of each arc in 30 min prior to infection. At time zero, DMEM with inhibitor or neutral 2 d gels were quantified (boxed areas in Figure 4C, D, E, solvent was removed, and fresh warm DMEM containing inhibitor F). Background signal in an area of equal size was subtracted, and or solvent and SV40 was added to cells. Cells were gently rocked the values for each arc were normalized to the value for the double every 15 min during the first 2 hpi. At 2 hpi, complete DMEM Y (Figure 4H) or bubble arc (Figure 4I). containing inhibitor or solvent was added to each dish of cells. At 20 hpi, medium was aspirated and cells were washed once with Statistics PBS to remove residual inhibitor or solvent. Fresh medium Statistics were performed in Microsoft Excel using the data containing inhibitor or solvent was then added to cells and analysis package. Prior to t-test, single factor ANOVA analysis was infections were allowed to proceed until the chosen endpoint. performed. If ANOVA resulted in p,0.5, a two sample t-test Solvent control treatments utilized the solvent concentration assuming unequal variances was performed. One-tailed p values present in the inhibitor-treated medium. from student’s t test are denoted by the number of asterisk(s): * p,0.05 ** p,0.01 *** p,0.001 **** p,0.0001. All one tailed p DNA isolation values were generated by comparing data from SV40 infection in Total intracellular DNA was prepared from infected and mock- the presence of inhibitor to that from SV40 infection in the infected cells. For each experiment, all samples were prepared presence of DMSO. Bar graphs present the average of 3 to 4 from an equal number of cells. Cell pellets were resuspended in independent experiments and error bars represent standard 0.4 ml of TE (10 mM Tris pH 8.0, 1 mM EDTA). SDS, RNase deviation. A, proteinase K, and Tris pH 7.5 were added to a final concentration of 0.4%, 0.2 mg/ml, 50 ug/ml and 100 mM, Supporting Information respectively, in a total volume of 0.5 ml. Following overnight Figure S1 Viral replication centers co-localize with host digestion at 37uC, each sample was extracted twice with Tris- DNA replication factors in SV40-infected BSC40 cells. A– saturated phenol (pH 7.9) and once with 24:1 chloroform: isoamyl E. Merged images of chromatin-bound Tag and the indicated host alcohol. DNA was precipitated with sodium acetate and ethanol. DNA replication factors from mock- or SV40-infected BSC40 cells DNA was allowed to dissolve in T0.1E (10 mM Tris pH 8.0, at 48 hpi. Top image for each replication protein is a mock- 0.1 mM EDTA) for 2 days, and then digested overnight at 37uC infected cell. The fluorescence intensity in arbitrary units (AU) with 40 U of SacI-HF and XbaI (both from New England along the line shown in the merged image is graphed in the right Biolabs). Digested DNA was re-precipitated and then dissolved in panel. Scale bars, 10 mm. 50 mL of T0.1E per 2.5610 cells. Equal volumes of DNA were (TIF) loaded on gels for southern blots unless otherwise indicated. Figure S2 Host DNA replication proteins co-localize Agarose gel electrophoresis with Tag in SV40-infected U2OS cells. A–D. Representative One-dimensional 0.7% agarose gels in 16 TAE were electro- images of chromatin-bound Tag and the indicated host DNA phoresed at 10 V/cm for 1.5 h. Neutral 2 d gel electrophoresis replication proteins from SV40-infected U2OS cells at 48 hpi. was performed as previously described [37] with the following The fluorescence intensity in arbitrary units (AU) along the line modifications. The first dimension of the gel was electrophoresed shown in the merged image is graphed in the right panel. Scale at 1 V/cm through a 0.4% 16TAE for 22 h. 16TAE was found bars, 10 mm. to enhance separation of D-loop arc (data not shown). The second (TIF) dimension was electrophoresed at 5.5 V/cm through a 1.1% 16 Figure S3 Aberrant DNA structures accumulate in TBE gel containing 0.5 ng/ml ethidium bromide for 5.5 h with ATM-inhibited SV40-infected U2OS cells. A. Total DNA circulation. extracted at 48 hpi from SV40-infected BSC40 cells treated with Ku-55933 during the indicated phases of infection, as in Figure 2A, Southern blotting analysis was analyzed by southern blot. Lanes 1–5: DNA digested with Southern blotting was performed using radiolabeled probes for XbaI and SacI. Lanes 6–10: DNA digested with BglI. B. Southern SV40 and BSC40 mitochondrial DNA as described [34]. A probe blot of DNA replicated in SV40-infected U2OS cells in the for human mitochondrial DNA was generated by PCR amplifi- presence of ATM inhibitor during the indicated phases of cation (primers: U2OS Mito-F ACG CGA TAG CAT TGC GAG infection. C. Quantification of SV40 signal in monomeric forms AC; U2OS Mito-R CTT TGG GGT TTG GTT GGT TCG), and the whole sample in each lane, normalized to the followed by random priming. Hybridized blots were visualized corresponding signals in the DMSO solvent lane as in panel B. using a Typhoon Trio laser scanning imager (GE Healthcare) and D and E. Fraction of signal in monomer forms (D) or in the quantified using ImageQuant 5.2 (GE Healthcare). indicated DNA structure (E) in DNA extracted at 48 hpi from cells Bands or arcs corresponding to each DNA structure of interest treated with Ku-55933 during the indicated phases of infection as were quantified and the value from a region of the blot without in panel B. Values in C–E represent the average of 3 to 4 signal, e.g. Mock for SV40 probe, was subtracted as background. independent experiments. To compare the level of a DNA structure after a given treatment (EPS) (e.g. DNA structure (% of Total DNA)), the total signals for the Figure S4 Caffeine inhibits ATM and ATR activities in DNA were summed, and the signal of a discrete DNA structure (e.g. form I monomer) were divided by the total signal in the lane SV40-infected BSC40 cells. A. BSC40 cells were treated with caffeine during the indicated phases of a 48 h SV40 infection. B (e.g. [form I monomer signal]/[total signal in the lane]). To quantify variations in replication between treatments, all SV40 and C. Western blots of cell lysates from SV40-infected BSC40 DNA signals were normalized using the respective mitochondrial cells exposed to caffeine as depicted in (A). DNA signal. Normalized signals were then divided by the (TIF) PLOS Pathogens | www.plospathogens.org 13 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure S5 ATM and ATR inhibition increases aberrant from infected U2OS cells treated with ATRi, normalized to the DNA product accumulation. A, E. Southern blots of total corresponding signals from infected cells treated with DMSO. D. DNA extracted from BSC40 (A) or U2OS (E) cells treated with Fraction of total SV40 signal in the indicated DNA structures in caffeine during phases of SV40 infection as described in Figure infected U2OS cells exposed to ATRi. Bars in graphs in C, D S4A. B, F. Quantification of signal in SV40 monomer forms or represent the average of 3 to 4 independent experiments. total DNA in caffeine-treated BSC40 (B) or U2OS (F) cells, (EPS) normalized to that in DMEM solvent. C, D, G, H. SV40 signal in Figure S9 ATR inhibition results in replication fork monomer forms (C and G) or aberrant DNA structures (D and H) stalling and breakage. A. Diagrams of replication intermedi- accumulated in caffeine-treated BSC40 (C and D) or U2OS (G ates on a simple Y arc produced when ATR is inhibited. Cleavage and H) cells, divided by the total SV40 DNA signal in respective sites are denoted as a colored vertical line: BglI (orange), BamHI lane. Bars in B–D and F–H represent the average of 3 to 4 (green). I. Replication begins at the origin and forks diverge independent experiments. bidirectionally to produce theta-form replication intermediates. II. (EPS) Both replisomes progress unless a replication block (red triangle) is Figure S6 DNA-PK activity is dispensable in unper- encountered, causing a fork to stall. III. The stalled replication fork turbed SV40 infection. A. Experimental scheme for treatment is closest to orange BglI site (viral origin of replication). The of BSC40 cells with DNA PK inhibitors during phases of a 48 h functional replisome continues replication and converges with the SV40 infection. B., C. Southern blots of DNA extracted from stalled replication fork. IV. One-sided DSB forms at the BSC40 cells treated as in A with Nu7026 (B) or Nu7441(C). D. replicating fork of the late Cairns intermediate shown in (III) as Quantification of SV40 replication products as in B, C. it approaches the stall site. V. Simple Y DNA structure generated (EPS) by BglI or BamHI digestion of the broken late Cairns intermediate shown in (IV). VI. Diagram of the predicted outcome of the simple Figure S7 ATRi inhibits ATR activity in SV40-infected Y shown in panel (V) after neutral 2 d gel electrophoresis and BSC40 cells. A. Exposure of SV40-infected BSC40 cells to ATRi southern blot analysis. The stall point on the simple Y arc (light during defined phases of a 48 h infection. B. WST-1 viability assay green circle) corresponds to the simple Y in panel (V). of SV40-infected BSC40 cells treated with ATRi as described in A. (EPS) Values were normalized to SV40-infected cells in the presence of DMSO. Error bars represent four independent experiments. C, D. Protocol S1 Supporting methods. Western blot of cell lysates from SV40-infected BSC40 cells (DOCX) exposed to ATRi as indicated. (EPS) Acknowledgments Figure S8 ATR is needed for efficient viral DNA We thank Dr. David Cortez for generously sharing inhibitors and advice, replication in U2OS cells. A. Southern blot analysis of total AstraZeneca for Ku-55933, Dr. E. Kremmer for anti-Cdc45, Dr. DNA from BSC40 cells treated with ATRi during the indicated Katherine Friedman, Dr. Heather Lorimer, and Dr. Bonita Brewer for advice on 2 d gels, the members of the Fanning lab for discussion, and phases of infection as in Figure S7A. Lanes 1–5: DNA digested Ashley Sowd for moral support. with XbaI and SacI. Lanes 6–10: DNA digested with BglI. An equal amount of unit length SV40 DNA was loaded in each lane Author Contributions using the data in figure 5C using an equal number of cells. B. Total DNA from SV40-infected U2OS cells treated with ATRi as Conceived and designed the experiments: GAS EF. Performed the in Figure S7A was analyzed by southern blotting. C. Quantifica- experiments: GAS NYL. Analyzed the data: GAS NYL EF. Wrote the tion of SV40 signal in total and monomeric SV40 DNA forms paper: GAS EF. References 1. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play 13. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, et al. (2011) with knives. Mol Cell 40: 179–204. Selective killing of ATM- or p53-deficient cancer cells through inhibition of 2. Chu WK, Hickson ID (2009) RecQ helicases: multifunctional genome ATR. Nat Chem Biol 7: 428–430. caretakers. Nat Rev Cancer 9: 644–654. 14. Weitzman MD, Lilley CE, Chaurushiya MS (2010) Genomes in conflict: maintaining genome integrity during virus infection. Annu Rev Microbiol 64: 3. Stracker TH, Petrini JH (2011) The MRE11 complex: starting from the ends. 61–81. Nat Rev Mol Cell Biol 12: 90–103. 15. Weitzman MD, Lilley CE, Chaurushiya MS (2011) Changing the ubiquitin 4. Meek K, Dang V, Lees-Miller SP (2008) DNA-PK: the means to justify the ends? landscape during viral manipulation of the DNA damage response. FEBS Lett Adv Immunol 99: 33–58. 585: 2897–2906. 5. Guo Z, Kozlov S, Lavin MF, Person MD, Paull TT (2010) ATM activation by 16. Weller SK (2010) Herpes simplex virus reorganizes the cellular DNA repair and oxidative stress. Science 330: 517–521. protein quality control machinery. PLoS Pathog 6: e1001105. 6. Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through 17. Lau A, Swinbank KM, Ahmed PS, Taylor DL, Jackson SP, et al. (2005) intermolecular autophosphorylation and dimer dissociation. Nature 421: 499– Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase. Nat Cell Biol 7: 493–500. 7. Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of 18. Moody CA, Laimins LA (2009) Human papillomaviruses activate the ATM RPA-ssDNA complexes. Science 300: 1542–1548. DNA damage pathway for viral genome amplification upon differentiation. 8. Cimprich KA, Cortez D (2008) ATR: an essential regulator of genome integrity. PLoS Pathog 5: e1000605. Nat Rev Mol Cell Biol 9: 616–627. 19. Sakakibara N, Mitra R, McBride AA (2011) The papillomavirus E1 helicase 9. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, et al. activates a cellular DNA damage response in viral replication foci. J Virol 85: (2007) ATM and ATR substrate analysis reveals extensive protein networks 8981–8995. responsive to DNA damage. Science 316: 1160–1166. 20. Wallace NA, Robinson K, Howie HL, Galloway DA (2012) HPV 5 and 8 E6 10. Brown EJ, Baltimore D (2000) ATR disruption leads to chromosomal abrogate ATR activity resulting in increased persistence of UVB induced DNA fragmentation and early embryonic lethality. Genes Dev 14: 397–402. damage. PLoS Pathog 8: e1002807. 11. Casper AM, Nghiem P, Arlt MF, Glover TW (2002) ATR regulates fragile site 21. Dahl J, You J, Benjamin TL (2005) Induction and utilization of an ATM stability. Cell 111: 779–789. signaling pathway by polyomavirus. J Virol 79: 13007–13017. 12. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, et al. (2004) 22. Jiang M, Zhao L, Gamez M, Imperiale MJ (2012) Roles of ATM and ATR- Identification and characterization of a novel and specific inhibitor of the ataxia- Mediated DNA Damage Responses during Lytic BK Polyomavirus Infection. telangiectasia mutated kinase ATM. Cancer Res 64: 9152–9159. PLoS Pathog 8: e1002898. PLOS Pathogens | www.plospathogens.org 14 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity 23. Bullock PA (1997) The initiation of simian virus 40 DNA replication in vitro. 49. Hashimoto Y, Puddu F, Costanzo V (2012) RAD51- and MRE11-dependent Crit Rev Biochem Mol Biol 32: 503–568. reassembly of uncoupled CMG helicase complex at collapsed replication forks. 24. Borowiec JA, Dean FB, Bullock PA, Hurwitz J (1990) Binding and unwinding– Nat Struct Mol Biol 19: 17–24. how T antigen engages the SV40 origin of DNA replication. Cell 60: 181–184. 50. Petermann E, Helleday T (2010) Pathways of mammalian replication fork 25. Waga S, Stillman B (1994) Anatomy of a DNA replication fork revealed by restart. Nat Rev Mol Cell Biol 11: 683–687. reconstitution of SV40 DNA replication in vitro. Nature 369: 207–212. 51. Munoz-Galvan S, Tous C, Blanco MG, Schwartz EK, Ehmsen KT, et al. (2012) 26. Sowd GA, Fanning E (2012) A Wolf in Sheep’s Clothing: SV40 Co-opts Host Distinct roles of Mus81, Yen1, Slx1-Slx4, and Rad1 nucleases in the repair of Genome Maintenance Proteins to Replicate Viral DNA. PLoS Pathog 8: replication-born double-strand breaks by sister chromatid exchange. Mol Cell e1002994. Biol 32: 1592–1603. 27. Fanning E, Zhao K (2009) SV40 DNA replication: from the A gene to a 52. Chang Y, Moore PS (2012) Merkel cell carcinoma: a virus-induced human nanomachine. Virology 384: 352–359. cancer. Annu Rev Pathol 7: 123–144. 28. Shi Y, Dodson GE, Shaikh S, Rundell K, Tibbetts RS (2005) Ataxia- 53. DeCaprio JA (2009) Does detection of Merkel cell polyomavirus in Merkel cell telangiectasia-mutated (ATM) is a T-antigen kinase that controls SV40 viral carcinoma provide prognostic information? J Natl Cancer Inst 101: 905–907. replication in vivo. J Biol Chem 280: 40195–40200. 54. DiMaio D, Liao JB (2006) Human papillomaviruses and cervical cancer. Adv 29. Zhao X, Madden-Fuentes RJ, Lou BX, Pipas JM, Gerhardt J, et al. (2008) Virus Res 66: 125–159. Ataxia telangiectasia-mutated damage-signaling kinase- and proteasome-depen- 55. Chia W, Rigby PW (1981) Fate of viral DNA in nonpermissive cells infected with dent destruction of Mre11-Rad50-Nbs1 subunits in Simian virus 40-infected simian virus 40. Proc Natl Acad Sci U S A 78: 6638–6642. primate cells. J Virol 82: 5316–5328. 56. Moarefi IF, Small D, Gilbert I, Hopfner M, Randall SK, et al. (1993) Mutation 30. Rohaly G, Korf K, Dehde S, Dornreiter I (2010) Simian virus 40 activates ATR- of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) Delta p53 signaling to override cell cycle and DNA replication control. J Virol large T antigen specifically blocks SV40 origin DNA unwinding. J Virol 67: 84: 10727–10747. 4992–5002. 31. Tang Q, Bell P, Tegtmeyer P, Maul GG (2000) Replication but not transcription 57. Schneider C, Weisshart K, Guarino LA, Dornreiter I, Fanning E (1994) Species- of simian virus 40 DNA is dependent on nuclear domain 10. J Virol 74: 9694– specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA. Mol Cell Biol 14: 3176– 32. Boichuk S, Hu L, Hein J, Gjoerup OV (2010) Multiple DNA damage signaling and repair pathways deregulated by simian virus 40 large T antigen. J Virol 84: 58. Weisshart K, Taneja P, Jenne A, Herbig U, Simmons DT, et al. (1999) Two 8007–8020. regions of simian virus 40 T antigen determine cooperativity of double-hexamer 33. Hein J, Boichuk S, Wu J, Cheng Y, Freire R, et al. (2009) Simian virus 40 large assembly on the viral origin of DNA replication and promote hexamer T antigen disrupts genome integrity and activates a DNA damage response via interactions during bidirectional origin DNA unwinding. J Virol 73: 2201–2211. Bub1 binding. J Virol 83: 117–127. 59. Yardimci H, Wang X, Loveland AB, Tappin I, Rudner DZ, et al. Bypass of a 34. Zhou B, Arnett DR, Yu X, Brewster A, Sowd GA, et al. (2012) Structural basis protein barrier by a replicative DNA helicase. Nature 492: 205–209. for the interaction of a hexameric replicative helicase with the regulatory subunit 60. Tapper DP, Anderson S, DePamphilis ML (1982) Distribution of replicating of human DNA polymerase alpha-primase. J Biol Chem 287: 26854–26866. simian virus 40 DNA in intact cells and its maturation in isolated nuclei. J Virol 35. Cohen GL, Wright PJ, DeLucia AL, Lewton BA, Anderson ME, et al. (1984) 41: 877–892. Critical spatial requirement within the origin of simian virus 40 DNA 61. Tapper DP, DePamphilis ML (1980) Preferred DNA sites are involved in the replication. J Virol 51: 91–96. arrest and initiation of DNA synthesis during replication of SV40 DNA. Cell 22: 36. Lobrich M, Shibata A, Beucher A, Fisher A, Ensminger M, et al. (2010) 97–108. gammaH2AX foci analysis for monitoring DNA double-strand break repair: 62. Tapper DP, Anderson S, DePamphilis ML (1979) Maturation of replicating strengths, limitations and optimization. Cell Cycle 9: 662–669. simian virus 40 DNA molecules in isolated nuclei by continued bidirectional 37. Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two- replication to the normal termination region. Biochim Biophys Acta 565: 84–97. dimensional agarose gel electrophoresis. Methods Enzymol 262: 613–627. 63. Casper AM, Durkin SG, Arlt MF, Glover TW (2004) Chromosomal instability 38. Preiser PR, Wilson RJ, Moore PW, McCready S, Hajibagheri MA, et al. (1996) at common fragile sites in Seckel syndrome. Am J Hum Genet 75: 654–660. Recombination associated with replication of malarial mitochondrial DNA. 64. Shuda M, Feng H, Kwun HJ, Rosen ST, Gjoerup O, et al. (2008) T antigen EMBO J 15: 684–693. mutations are a human tumor-specific signature for Merkel cell polyomavirus. 39. Backert S (2002) R-loop-dependent rolling-circle replication and a new model Proc Natl Acad Sci U S A 105: 16272–16277. for DNA concatemer resolution by mitochondrial plasmid mp1. EMBO J 21: 65. Gjoerup O, Chang Y (2010) Update on human polyomaviruses and cancer. Adv 3128–3136. Cancer Res 106: 1–51. 40. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, et al. (1999) Inhibition of 66. An P, Saenz Robles MT, Pipas JM (2012) Large T antigens of polyomaviruses: ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer amazing molecular machines. Annu Rev Microbiol 66: 213–236. Res 59: 4375–4382. 67. Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, et al. (1985) 41. Chen BP, Chan DW, Kobayashi J, Burma S, Asaithamby A, et al. (2005) Cell Structure and transcription of human papillomavirus sequences in cervical cycle dependence of DNA-dependent protein kinase phosphorylation in carcinoma cells. Nature 314: 111–114. response to DNA double strand breaks. J Biol Chem 280: 14709–14715. 68. Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M (2009) Mechanism of 42. Pohlhaus JR, Kreuzer KN (2006) Formation and processing of stalled replication genomic instability in cells infected with the high-risk human papillomaviruses. forks–utility of two-dimensional agarose gels. Methods Enzymol 409: 477–493. PLoS Pathog 5: e1000397. 43. Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, et al. (2001) The 69. Woodman CB, Collins SI, Young LS (2007) The natural history of cervical HPV DNA replication checkpoint response stabilizes stalled replication forks. Nature infection: unresolved issues. Nat Rev Cancer 7: 11–22. 412: 557–561. 70. Hardcastle IR, Cockcroft X, Curtin NJ, El-Murr MD, Leahy JJ, et al. (2005) 44. Lukas J, Lukas C, Bartek J (2011) More than just a focus: The chromatin Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein response to DNA damage and its role in genome integrity maintenance. Nat Cell kinase (DNA-PK) using a small-molecule library approach. J Med Chem 48: Biol 13: 1161–1169. 7829–7846. 45. Rigby PW, Berg P (1978) Does simian virus 40 DNA integrate into cellular DNA 71. Leahy JJ, Golding BT, Griffin RJ, Hardcastle IR, Richardson C, et al. (2004) during productive infection? J Virol 28: 475–489. 46. Ilves I, Tamberg N, Botchan MR (2012) Checkpoint kinase 2 (Chk2) inhibits the Identification of a highly potent and selective DNA-dependent protein kinase activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex. (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Proc Natl Acad Sci U S A 109: 13163–13170. Med Chem Lett 14: 6083–6087. 47. Liu P, Barkley LR, Day T, Bi X, Slater DM, et al. (2006) The Chk1-mediated S- 72. Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW (2003) phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2- Radiosensitization and DNA repair inhibition by the combined use of novel independent mechanism. J Biol Chem 281: 30631–30644. inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase- 48. Lydeard JR, Lipkin-Moore Z, Sheu YJ, Stillman B, Burgers PM, et al. (2010) 1. Cancer Res 63: 6008–6015. Break-induced replication requires all essential DNA replication factors except 73. Hirt B (1967) Selective extraction of polyoma DNA from infected mouse cell those specific for pre-RC assembly. Genes Dev 24: 1133–1144. cultures. J Mol Biol 26: 365–369. PLOS Pathogens | www.plospathogens.org 15 April 2013 | Volume 9 | Issue 4 | e1003283 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png PLoS Pathogens Public Library of Science (PLoS) Journal

ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication

PLoS Pathogens , Volume 9 (4): e1003283 – Apr 4, 2013
Download PDF
15 pages

Loading next page...
 
/lp/public-library-of-science-plos-journal/atm-and-atr-activities-maintain-replication-fork-integrity-during-sv40-nhWZTc93JW

References (152)

Publisher
Public Library of Science (PLoS) Journal
Copyright
Copyright: © 2013 Sowd et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants (GM52948 to EF, T32 AI089554 to JE Crowe, P30 CA068485 to the Vanderbilt-Ingram Cancer Center), and Vanderbilt University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.
Subject
Research Article; Biology; Biochemistry; Nucleic acids; DNA; DNA recombination; DNA repair; DNA replication; Virology; Biochemistry
ISSN
1553-7366
eISSN
1553-7374
DOI
10.1371/journal.ppat.1003283
Publisher site
See Article on Publisher Site

Abstract

Mutation of DNA damage checkpoint signaling kinases ataxia telangiectasia-mutated (ATM) or ATM- and Rad3-related (ATR) results in genomic instability disorders. However, it is not well understood how the instability observed in these syndromes relates to DNA replication/repair defects and failed checkpoint control of cell cycling. As a simple model to address this question, we have studied SV40 chromatin replication in infected cells in the presence of inhibitors of ATM and ATR activities. Two-dimensional gel electrophoresis and southern blotting of SV40 chromatin replication products reveal that ATM activity prevents accumulation of unidirectional replication products, implying that ATM promotes repair of replication-associated double strand breaks. ATR activity alleviates breakage of a functional fork as it converges with a stalled fork. The results suggest that during SV40 chromatin replication, endogenous replication stress activates ATM and ATR signaling, orchestrating the assembly of genome maintenance machinery on viral replication intermediates. Citation: Sowd GA, Li NY, Fanning E (2013) ATM and ATR Activities Maintain Replication Fork Integrity during SV40 Chromatin Replication. PLoS Pathog 9(4): e1003283. doi:10.1371/journal.ppat.1003283 Editor: Michael J. Imperiale, University of Michigan, United States of America Received November 27, 2012; Accepted February 14, 2013; Published April 4, 2013 Copyright:  2013 Sowd et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by NIH grants (GM52948 to EF, T32 AI089554 to JE Crowe, P30 CA068485 to the Vanderbilt-Ingram Cancer Center), and Vanderbilt University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: [email protected] Interestingly, multiple animal viruses have evolved to manipu- Introduction late DNA damage signaling pathways to facilitate viral propaga- Faithful duplication of the genome is vital for cell proliferation. tion [14]. Some viruses, e.g. Herpes simplex, evade or disable In metazoans, the consequences of inaccurate genome replication DNA damage response pathways that result in inappropriate include cell death, premature aging syndromes, neuro-degenera- processing of viral DNA [15,16]. In other cases, viral infection tion disorders, and susceptibility to cancer [1,2]. The DNA appears to activate checkpoint signaling and harness it to promote damage signaling protein kinases ataxia telangiectasia-mutated the infection. HIV, human papillomaviruses, and polyomaviruses (ATM) and ATM- and Rad3-related kinase (ATR), members of induce and depend on ATM signaling for viral propagation the phosphoinositide-3 kinase-like kinase (PIKK) family, act to [17,18,19,20,21,22]. However, mechanistic understanding of how ensure that cells with incompletely replicated or damaged DNA do these viruses activate damage signaling and exploit it for viral not progress through the cell cycle [1]. ATM and DNA-dependent propagation is limited. protein kinase (DNA-PK) respond primarily to DNA double Simian Virus 40 (SV40), a polyomavirus that propagates in strand breaks (DSB) that are associated with either Mre11/NBS1/ monkey kidney cells, has served as a powerful model to study Rad50 (MRN) [3] or Ku70/80 [4], respectively. Additionally, eukaryotic replication proteins and mechanisms in vivo and in vitro intracellular oxidation or alterations in chromatin structure can [23,24,25,26,27]. Checkpoint signaling proteins are dispensable for activate ATM kinase [5,6]. In contrast, single-stranded DNA SV40 DNA replication in vitro, yet in infected cells, ATM or ATR (ssDNA) bound by RPA activate ATR [7,8]. When activated, knockdown, over-expression of kinase-dead variant proteins, or ATM and ATR phosphorylate consensus SQ/TQ motifs in target chemical inhibition of checkpoint signaling clearly decreases or proteins at sites of damage, e.g. the histone H2AX, which delays SV40 chromatin replication [26,28,29,30]. To determine facilitates recruitment of repair proteins and activation of how checkpoint signaling facilitates viral replication in SV40- downstream kinases Chk1 and Chk2 that enforce the checkpoint [8,9]. infected primate cells, we have utilized small molecule inhibitors of the PIKK family members ATM, ATR, and DNA-PK to suppress Failure to activate DNA damage checkpoints results in genome instability syndromes. Mutations in the human ATM gene can checkpoint signaling in host cells during three specific time windows cause the cancer-prone disorder ataxia telangiectasia. Hypomor- after SV40 infection. Characterization of the resulting viral DNA replication products reveals that inhibition of ATM or ATR, but not phic mutations in the ATR gene can cause the genomic instability disorder Seckel Syndrome, but complete loss of ATR results in cell DNA-PK, reduced the yield of unit length viral replication products and caused aberrant viral DNA species to accumulate. ATM death [10,11]. The central roles of ATM and ATR in genome maintenance suggest the potential to manipulate their activity for inhibition led to unidirectional SV40 DNA replication and cancer chemotherapy, fueling the development of potent small concatemeric products, whereas ATR inhibition markedly in- molecules that specifically inhibit ATM and ATR activities in creased broken SV40 DNA replication forks. Our results strongly cellulo [12,13]. suggest that unperturbed viral chromatin replication in infected cells PLOS Pathogens | www.plospathogens.org 1 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity We next asked whether SV40 DNA replication itself might Author Summary induce DNA damage signaling in the absence of viral infection. All cells have evolved pathways to maintain the integrity of Toward this end, the plasmids pMini SV40-wt, and its replication- the genetic information stored in their chromosomes. defective variants lacking Tag helicase activity (D474N) [34], or Endogenous and exogenous agents induce mutations and containing a single base pair insertion that inactivates the viral other damage in DNA, most frequently during DNA origin (In-1) [35], were transfected into BSC40 monkey cells replication. Such DNA damage is under surveillance by a (Figure 1B). As expected, all three plasmids expressed Tag, but complex network of proteins that interact with one only the SV40-wt plasmid replicated (Figure 1C, D). SV40-wt another to signal damage, arrest DNA replication, and activated phosphorylation of Chk1 and Chk2 more robustly than restore genomic integrity before replication resumes. either of the replication-defective constructs (Figure 1C, compare Many viruses that replicate in the nucleus of mammalian lane 1 to lanes 2–3). Moreover, prominent cH2AX foci, a marker host cells have evolved to disable or evade this surveil- of DNA damage signaling in chromatin [36], colocalized with lance system, but others, e.g. polyomaviruses like SV40, chromatin-bound Tag in viral replication centers in SV40-wt activate it and somehow harness it to facilitate robust transfected cells (Figure 1E). In contrast, the few cH2AX foci replication of viral progeny. We have sought to determine detected in cells transfected with the replication defective plasmids how SV40 induces and deploys host DNA damage did not colocalize with Tag. Thus, in the context of transfected signaling in infected cells to promote viral chromosome cells, viral DNA replication, but not SV40-driven Tag expression, replication. Here we present evidence that, like host DNA, is sufficient to induce DNA damage signaling, suggesting that replicating viral DNA suffers damage that activates DNA breaks in replicating viral chromatin may activate check- surveillance and repair pathways. Unlike host replication, point signaling. viral DNA replication persists despite damage signaling, allowing defective replication products to accumulate. In the presence of host DNA damage signaling, these Inhibition of ATM disrupts viral DNA replication centers defective viral products attract proteins of the host To determine the temporal requirements for ATM activity damage surveillance network that correct the defects, during infection, we exposed infected cells to the specific ATM thus maximizing viral propagation. chemical inhibitor Ku-55933 [12] during the early phase (virus entry, Tag expression, host DNA synthesis), late phase (viral DNA results in double strand breaks, activating checkpoint signaling and replication, late gene expression, and virion assembly), or fork repair to generate unit length viral replication products. throughout a 48-hour infection (Figure 2A). Infected cells exposed to the Ku-55933 solvent, DMSO, served as a positive control. Mock-infected cells not treated with inhibitor served as a negative Results control. ATM activity was stimulated by infection, as indicated by SV40 chromatin replication activates DNA damage phosphorylated Nbs1 and Chk2 in western blots (Figure 2B, compare lane 1 to lane 5), reduced by the presence of Ku-55933 in signaling either the early or late phase of infection (Figure 2B, compare Replicating SV40 chromatin in infected cells has been lanes 2, 3 to lane 1), and nearly abolished by the presence of Ku- visualized by fluorescence microscopy in prominent subnuclear 55933 throughout infection (Figure 2B, lane 4). foci that co-localize with Tag and several host proteins essential for To assess the impact of ATM inhibition during each phase of viral DNA replication in vitro, suggesting that these foci may infection on viral chromatin replication, we visualized viral represent viral chromatin replication centers [26,29,31]. However, replication centers and DNA damage signaling in each infected SV40 infection activates ATM and ATR signaling, and several cell population using immunofluorescence microscopy (Figure 2C). DNA damage signaling proteins, e.g. MRN, cH2AX, ATRIP, and In infected cells exposed to DMSO, the normal, brightly stained 53BP1, co-localize with Tag in these foci [28,29,30,32], implying a viral replication centers with colocalized Tag, EdU, and cH2AX link between SV40 replication and damage signaling. On the were observed (Figure 2C). When Ku-55933 was present only other hand, interaction of ectopically expressed Tag with the during the early phase of infection, about half of the cells displayed spindle checkpoint protein Bub1 can also induce cellular normal replication centers with colocalized Tag, EdU and cH2AX chromosome breaks [33], indicating that Tag interference with foci (Figure 2C and D). However, aberrant pan-nuclear staining of host mitotic checkpoint proteins may suffice to damage genomic Tag, EdU, and cH2AX predominated when Ku-55933 was DNA in uninfected cells. present during the late phase or throughout infection (Figure 2C As a first step to assess a potential link between SV40 chromatin and D). Taken together, the results demonstrate that ATM activity replication and DNA damage signaling, viral replication centers in was beneficial but not essential during the early phase of infection, SV40-infected BSC40 monkey cells were characterized in detail. whereas it was vital for the assembly and/or stability of viral Chromatin-bound Tag was visualized in subnuclear foci as replication centers during the late phase of infection. expected and colocalized with newly replicated DNA that had incorporated the deoxynucleoside EdU (Figures 1A and S1A). Inhibition of ATM activity reduces the quantity and Chromatin-bound PCNA, DNA polymerase d, and the clamp- loader RFC, host proteins that are essential for viral DNA quality of viral replication products replication in vitro, colocalized with Tag foci in both BSC40 and The links between ATM activity and SV40 replication centers led human U2OS cells at 48 hours post infection (hpi) (Figures 1A, us to hypothesize that inhibition of ATM might affect not only the S1B–D, and S2). In contrast, Cdc45, an essential component of level, but perhaps also the nature of the viral DNA replication the CMG host replicative helicase that colocalized with replicating products. To investigate this possibility, we used southern blotting to chromatin in mock-infected U2OS cells (Figure S2C, D), was analyze total intracellular DNA from SV40-infected BSC40 cells virtually excluded from viral replication centers (Figures 1A, S1E, that had been treated with DMSO or Ku-55933 throughout and S2C, D). The results strongly suggest that in infected cells, infection (Figure 3A). Inhibition of ATM reduced the level of 5.2 these chromatin-bound Tag foci represent sites of viral, rather kbp viral DNA products migrating as form I (supercoiled), form II than host, chromatin replication. (nicked), and form III (linear), relative to that in the DMSO-treated PLOS Pathogens | www.plospathogens.org 2 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 1. SV40 chromatin replication results in DNA damage signaling. (A) Representative images of chromatin-bound Tag and host DNA replication proteins in SV40- and mock-infected (inset) BSC40 cells at 48 hpi. (B) Features of the SV40 genome and the insertion site of pMini vector [34]. Mutation of Tag residue 474 from D to N abrogates helicase activity [34]. The defective SV40 origin mutant, In-1, features an insertion of a single PLOS Pathogens | www.plospathogens.org 3 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity GC bp in the center of the viral origin allowing Tag binding, but not origin activation [35]. (C, D, E) BSC40 cells transfected with the indicated pMini SV40 plasmids were analyzed by (C) western blot after 24 h, (D) Southern blot of low molecular weight DNA after 48 h [34,73], or (E) immunofluorescence microscopy of chromatin-bound proteins. In (D), SV40 or Mitochondrial probe signal is denoted by SV40 or Mito, respectively. Scale bars in (A) and (E), 10 mm. doi:10.1371/journal.ppat.1003283.g001 control infections (Figure 3A, compare lanes 1–4 to 5–8). However, To quantify the data in Figure 3A, the signal in SV40 monomer ATM inhibition also caused accumulation of high molecular weight bands (forms I, II, and III) in each sample was normalized to that SV40 DNA products too large to enter the gel (Figure 3A, compare of mitochondrial DNA (Mito) in the same sample. This lanes 3, 4 to lanes 7, 8). These large products failed to migrate into normalized monomer signal in each sample was then compared the gel after restriction digestion with enzymes that cut host DNA to that of the normalized monomer bands in the positive control at but not SV40 DNA. In contrast, most of these products collapsed 72 hpi. (Figure 3A, lane 4) and graphed in Figure 3B. The graph into unit length linear SV40 DNA after digestion with an enzyme reveals that ATM inhibition reduced unit length SV40 product by that cleaves SV40 DNA once (Figure S3A), indicating that the large at least 5-fold compared to the DMSO control infections DNA products contain head-to-tail repeats of unit length viral (Figure 3B). Quantification of the concatemeric SV40 DNA in DNA. each sample relative to that of the total SV40 signal in the same Figure 2. ATM inhibition during viral DNA replication disrupts viral replication centers. (A) Experimental scheme for treatment of cells with inhibitor during phases of a 48 h SV40 infection. Early: inhibitor present from 20.5 to 20 hpi. Late: inhibitor present from 20 to 48 hpi. DMSO and Full: solvent or inhibitor, respectively, present from 20.5 to 48 hpi. (B) Western blot of cells treated with Ku-55933 as described in (A). (C) Immunofluorescence of cells treated with Ku-55933 as described in (A) and fixed at 48 hpi. Scale bars, 10 mm. (D) Tag staining patterns, as in (C), were quantified. Graph shows the average of 3 independent experiments. doi:10.1371/journal.ppat.1003283.g002 PLOS Pathogens | www.plospathogens.org 4 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity PLOS Pathogens | www.plospathogens.org 5 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 3. Ku-55933 treatment during viral DNA replication increases aberrant DNA structures. (A) Southern blot of DNA from SV40 infected BSC40 cells in the presence of DMSO or Ku-55933. DMSO or Ku-55933 was present from 30 min prior to infection until cell collection timepoint. M represents Mock-infected cells. (B) Each normalized monomer SV40 form I, II, and III product in (A) was graphed as a fraction of the corresponding normalized monomer produced at 72 hpi in the DMSO control infection. (C) Graph of the percentage of DNA products represented by concatemers in panel (A). (D) Southern blot of SV40 DNA replicated in the presence of Ku-55933 during phases of a 48 h infection in BSC40 cells as explained in Figure 2A. (E) Quantification of total and monomeric SV40 DNA signal normalized to DMSO control from southern blots as in (D). (F, G) Graph of DNA structures (monomer: F and DNA Structure: G) accumulating on southern blots as in (D). Graphs in (E–G) represent 3 to 4 independent experiments. doi:10.1371/journal.ppat.1003283.g003 sample revealed that ATM inhibition increased accumulation of the bubble arc was absent and the unit-mass viral DNA migrated in the 1 n spot as expected (Figure 4A–C). Also as expected, an viral DNA concatemers by an order of magnitude compared to that in the DMSO control samples (Figure 3C). Thus, inhibition of intense double Y arc indicative of converging forks and an X structure signal indicative of hemi-catenates or Holliday junctions ATM throughout infection reduced monomeric and increased were observed (Figure 4C). In addition, the simple Y arc signal concatemeric SV40 DNA products. revealed some unidirectional replicating forks (Figure 4C) that can To determine what stage of SV40 infection required ATM be most easily explained by rolling circle replication. When activity, total intracellular DNA was extracted from infected BamHI-cleaved DNA from DMSO-treated infected cells was BSC40 cells exposed to Ku-55933 during three time windows, as analyzed by 2 d gel electrophoresis, the bubble arc was detected diagrammed in Figure 2A. The purified DNA was separated by and the double Y arc was absent, as expected (Figure 4D). Similar gel electrophoresis and analyzed in southern blots (Figure 3D). to BglI digestion, both an X structure and a weaker simple Y arc Inhibition of ATM either early or throughout infection reproduc- were present (Figure 4D). ibly reduced the level of total viral DNA and monomeric DNA In contrast, the pattern of BglI-digested viral replication products by 50–80% relative to that generated in the DMSO- intermediates generated in the presence of Ku-55933 displayed treated control infection (Figure 3D, E). Similarly, in the late phase a much fainter double Y arc and a more intense simple Y arc of infection, inhibition reduced viral DNA monomers to a level (compare Figure 4E with C). Similarly, X structures and D-loops, comparable to that observed when ATM was inhibited during the or other complex branched intermediates (red star), were more early phase, yet total viral DNA was only insignificantly decreased prominent when ATM was inhibited (compare Figure 4E with C), compared to DMSO-treated cells (Figure 3D, E). SV40 monomers consistent with increased Holliday junction formation between comprised about 80% of the total viral DNA signal in samples replicating rolling circles [38,39]. Likewise, BamHI-cleaved from infected cells exposed to DMSO or Ku-55933 during early replication intermediates from Ku-55933-treated infections dis- phase (Figure 3F). In contrast, monomers comprised only 64% of played a robust simple Y arc and a corresponding decrease in the the total signal in samples treated with Ku-55933 late or bubble arc (Figure 4F). Moreover, the intense X structure and D- throughout infection (Figure 3F). When Ku-55933 was applied loop arcs were retained (Figure 4F). These patterns suggest that either during the late phase or throughout infection, the fraction of inhibition of ATM sharply increased the frequency of rolling circle total viral DNA in concatemers increased 10- and 11-fold, replication (Figure 4G). Quantification of the signal present in the respectively, relative to the fraction in DMSO-treated infected simple Y, X structure, D-loop, and double Y arcs from BglI- cells (Figure 3G). The fraction of total SV40 DNA migrating at 20- digested DNA (Figure 4C, E boxes) showed that ATM inhibition kbp linear also increased in cells treated with Ku-55933 late or increased the abundance of simple Ys, X structures, and D-loop throughout infection, relative to that in DMSO-treated control arcs relative to the double Y arc by six, three, and eight-fold, infections (Figure 3G). respectively, from three to four independent experiments To confirm these findings in a different cell background, the (Figure 4H). Analogously, quantification of BamHI-digested temporal requirements for ATM activity were also determined in DNA (Figure 4D, F boxes) revealed ATM inhibition increased SV40-infected human U2OS cells, with similar results (Figure the quantities of simple Ys, X structures, and D-loop arcs relative S3B–E). Taking the results together, we infer that SV40-infected to the bubble arc (Figure 4I). We conclude that the ATM inhibitor cells require ATM signaling, primarily during the late phase of Ku-55933 increased both rolling circle replication and strand infection, to favor production of unit-length genomes rather than invasion events at the expense of bidirectional SV40 chromatin aberrant products. replication. ATM inhibition increases rolling circle DNA replication Caffeine inhibits SV40 chromatin replication and strand invasion The importance of ATM activity in SV40 chromatin replication To better understand how the aberrant viral replication suggested the possibility that other checkpoint kinases might also products arise, we compared replication intermediates generated contribute to viral infection. To further explore this question, we with and without Ku-55933 during the late phase of infection. The treated SV40-infected BSC40 cells with caffeine, a less selective total DNA was first digested with a restriction nuclease that cleaves inhibitor of both ATM and ATR in vitro and of the S/G2 SV40 once in the viral origin (BglI) or once in the region of checkpoints in vivo [40]. Of note, caffeine is structurally unrelated termination (BamHI). Neutral two-dimensional (2 d) gel electro- to the more potent Ku-55933 and ATR inhibitors [12,13]. As phoresis was then used to separate viral replication intermediates expected, caffeine inhibited phosphorylation of Chk1 and Chk2 from the accumulated non-replicating unit-mass SV40 DNA, when present during the late phase or throughout infection (Figure followed by southern blotting using the whole SV40 genome as the S4A, B) but also hyper-activated DNA-PK (Figure S4B, compare probe [37]. Replicating viral DNA is present in the form of lane 1 with lanes 2–4) [41]. Caffeine reduced the level of total viral circular, converging forks known as Cairns intermediates DNA products in SV40-infected BSC40 cells to less than 1% of (Figure 4B). The digestion of Cairns intermediates with BglI or the control level when caffeine was present throughout infection BamHI results in double Ys or bubbles, respectively (Figure 4A, B). (Figure S5A, B). Exposure to caffeine late or throughout infection In the BglI-cleaved DNA from DMSO-treated control infections, reduced the fraction of total viral DNA signal in monomers (form PLOS Pathogens | www.plospathogens.org 6 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity PLOS Pathogens | www.plospathogens.org 7 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 4. ATM inhibition increases recombination and unidirectional replication. (A) Diagram of neutral 2 d gel electrophoresis arcs generated from digested SV40 DNA. (B) Replicating viral DNA extracted from unperturbed SV40-infected cells consists primarily of circular, late replication intermediates called late Cairns intermediates. Digestion of late Cairns intermediates with BglI yields large double Ys, whereas BamHI digestion yields large bubbles. (C, D, E, F) Southern blot of neutral 2 d gel of BglI-cleaved DNA replicated in the presence of DMSO (C, D) or Ku-55933 (E, F) during the late phase of a 48 h SV40 infection in BSC40 cells. DNA was cleaved within the viral origin of replication with BglI (C, E) or the region of fork convergence with BamHI (D, F). The red star denotes an arc representing strand invasion events (D-loops) or highly branched molecules [38,39]. On the simple Y arc in (F), S denotes a replication stall point near the viral origin of replication. Dashed boxes denote regions of each arc quantified in (H) and (I). (G) Concatemers of SV40 DNA that accumulated when ATM was inhibited can arise by either replication- (top) or recombination- (bottom) dependent rolling circle replication. Digestion of replication-dependent rolling circles with BglI or BamHI results in simple Ys of all sizes. Digestion of recombination-dependent rolling circles creates D-loops of all sizes. (H) Graph of DNA signal present on simple Y, double Y,X structure, or D-loop arc divided by DNA signal in the double Y arc from DNA digested with BglI. (I) Graph of DNA signal from BamHI digested DNA in simple Y, bubble, X structure, or D-loop arc divided by DNA signaling in the bubble arc. Each graph in (H) and (I) represents the average of 3 to 4 independent experiments. doi:10.1371/journal.ppat.1003283.g004 I, II, III) and increased the fraction in concatemers and other S8B–D). Taken together, these results indicate that infected cells aberrant products (Figure S5A, C, D). Similarly, in SV40-infected require ATR activity before, as well as during viral chromatin U2OS cells, caffeine reduced total viral replication products and replication, for normal accumulation of viral genomes. increased the fraction of aberrant products (Figure S5E–H). The results further confirm the role of ATM activity in SV40 Broken and/or stalled forks accumulate in ATR-inhibited chromatin replication in infected cells and suggest that ATR SV40-infected cells and/or DNA-PK activity may stimulate viral replication. The structures of viral replication intermediates generated in the presence and absence of ATR kinase activity were characterized DNA-PK activity is dispensable for SV40 chromatin by using neutral 2 d gel electrophoresis and southern blotting. As replication expected, BglI-digested SV40 replication intermediates from Although SV40 infection did not activate DNA-PK, it was control infections displayed a strong double Y arc indicative of activated in infected cells exposed to Ku-55933 or caffeine, as converging forks, X structures, and a weaker simple Y arc with evidenced by DNA break-dependent auto-phosphorylation of both legs of similar intensity (Figure 6B). In contrast, BglI-digested DNA-PK at S2056 [41] (Figures 2B, S4B). To test for a potential replication intermediates from ATRi-treated cells yielded a novel role of DNA-PK activity in viral chromatin replication, SV40- pattern (Figure 6C). Although the double Y and X structure arcs infected BSC40 cells were exposed to small molecule inhibitors of closely resembled those in the DMSO control, the simple Y arc DNA-PK during the early or late phase, or throughout infection displayed much greater intensity in the leg closer to the 1 n linear and total intracellular DNA was analyzed by southern blotting DNA (Figure 6B and C, zoomed box) than in the other leg closer (Figure S6A–C). When DNA-PK was inhibited with either to 2 n linear DNA. This pattern is not consistent with rolling circle Nu7441 or Nu7026, the levels of viral monomer and aberrant replication, which generates a uniformly intense simple Y arc viral DNA products closely resembled those in SV40-infected (Figure 4) or with two stalled replication forks, of which one BSC40 cells (Figure S6D). Moreover, inhibition of DNA-PK had breaks, creating an asymmetric simple Y [42]. The observed little or no effect on viral replication centers (data not shown). pattern is also inconsistent with one normal replication fork and Thus, it is unlikely that DNA-PK has a major role in viral one slower moving fork, which would converge asymmetrically to chromatin replication in unperturbed infected cells. generate a cone-shaped signal between the X structure arc and the Y arc [43]. However, the novel pattern observed could arise if one ATR inhibition decreases SV40 DNA replication fork stalls prematurely (Figure 6F, I, II), while the other fork The role of ATR kinase activity in infection was directly progresses until it encounters the stalled fork and then breaks, examined by treating SV40-infected BSC40 cells with a specific generating a broken late Cairns intermediate (Figure 6F, III, IV) small molecule inhibitor of ATR, VE-821 (ATRi) [13], during [37]. Close inspection of the intense leg of the Y arc reveals that its three different time windows of infection (Figure S7A). As intensity is uneven, suggesting that it may arise from a series of expected, ATRi caused a third of the cells to lose viability over closely spaced break sites along the Y arc (Figure 6C). If the break 48 h, but SV40-infected and mock-infected cells were equally sites reside 2.5 kb or less from the BglI cleavage site, the intensity sensitive (Figure S7B). SV40 infection activated Chk1, as indicated of signals would be greater in the right leg of the simple Y arc, as by phosphorylation of Ser317 (Figure S7C, compare lane 1 with observed (Figure 6C, box). This interpretation predicts that if lane 5), and ATRi effectively suppressed ATR activation during replication products from the ATRi-treated infection were each time window (Figure S7C, lanes 2–4). digested with BamHI, which cleaves 2.5 kb from the BglI site, Viral DNA replication products from the four cell populations the sites of breakage, and hence greater signal intensity, should and mock-infected cells were analyzed by southern blotting and shift to the left leg of the simple Y arc, closer to the 2 n linear DNA quantified relative to mitochondrial DNA in the same samples. In (Figure 6A, E). Indeed, this shift was observed (compare Figure 6D the presence of ATRi, the level of total viral DNA replication with E), confirming that when the moving replication fork products declined markedly relative to that in DMSO-treated encountered a fork that had stalled in the presence of ATRi, the control infections, amounting to only 10% of the control when moving fork broke (Figures 6F and S9). ATRi was present for the full 48 h (Figure 5B, C). In cells exposed to ATRi during the late phase or throughout infection, the fraction Discussion of viral DNA products in monomers (forms I, II and III) dropped, whereas that in concatemers and other aberrant products rose This study presents several lines of evidence that SV40 (Figure 5B–E and Figure S8A). Analysis of viral replication harnesses host DNA damage signaling for quality control of viral products from SV40-infected U2OS cells exposed to ATRi chromatin replication. We show that viral DNA replication in vivo demonstrated a similar requirement for ATR activity (Figure is sufficient to induce DNA damage signaling at viral replication PLOS Pathogens | www.plospathogens.org 8 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 5. ATR is crucial for SV40 chromatin replication. (A) Scheme for application of ATRi during phases of a 48 h SV40 infection. (B) Southern blot of DNA replicated in BSC40 cells when ATRi was present during phases of a 48 h SV40 infection described in (A). (C) Graph of total viral or SV40 monomer DNA signals normalized to SV40 DNA replicated in the presence of DMSO from southern blots as shown in (B). (D, E) Graph of of monomer (D) or aberrant (E) structure(s) accumulated as a result of ATR inhibition from southern blots as shown in (C). Each bar in (C–E) shows the average from 3 to 4 independent experiments. doi:10.1371/journal.ppat.1003283.g005 centers (Figures 1, S1, S2), suggesting that DNA lesions may arise DNA damage signaling nucleates the assembly of SV40 in unperturbed replicating viral DNA. Importantly, damage replication centers signaling is vital to maintain viral replication centers (Figures 1, SV40 chromatin replication centers resemble over-sized host 2). Furthermore, suppression of ATM and/or ATR signaling DNA damage response foci (for a comparison, see Figure 1 in ref increases the level of aberrant viral replication products at the [29]), where diverse damage signaling and DNA repair proteins expense of unit length viral DNA (Figures 3–5, S3, S5, S8), assemble on chromatin at a DNA lesion and dissociate when implying that viral replication-associated damage in infected cells repair is completed [1,44]. Many of the same signaling and repair requires ATM and ATR signaling to promote repair of viral proteins are found at both viral replication centers and host replication forks. Lastly, our results indicate that the defective damage response foci [18,21,22,28,29,30,32,33] (Sowd, unpub- replication intermediates resulting from inhibition of ATM lished). However, unlike the prominent viral replication centers, (Figure 4) and ATR (Figures 6, S9) are distinctive. Taken together, the punctate host damage response foci encompass megabase our results support a model in which ATM and ATR serve regions of chromatin, raising the question of how SV40 mini- different but complementary roles in orchestrating repair at viral chromosomes give rise to the large subnuclear foci observed in the replication forks (Figure 7). microscope. The size of SV40 replication centers increases with PLOS Pathogens | www.plospathogens.org 9 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 6. ATR inhibition results in fork stalling and breakage of converging forks. (A) Schematic of replication intermediate migration patter on a neutral 2 d gel generated from digested SV40 DNA. (B, C, D, E) Southern blot of neutral 2 d gel electrophoresis of BglI- (B, C) or BamHI-cut (D, E) DNA from SV40-infected BSC40 cells exposed to DMSO (B, D) or ATRi (C, E) during the late phase of SV40 infection as described in Figure 5A. (F) Diagrams of replication intermediates on a simple Y arc produced when ATR was inhibited. BamHI (green) and BglI (orange) sites are denoted by colored lines. I. Replication initiates at the origin and proceeds bidirectionally producing theta replication intermediates. II. Replisomes continue replication until one encounters a replication block (red triangle) causing one stalled fork. III. The stalled replication fork is closest to orange BglI site (viral origin of replication). The functional replisome continues replication and converges with the stalled replication fork. IV. One-sided DSB forms at the replicating fork of late Cairns intermediate shown in (III) as it translocates toward the stall site. V. Simple Y created by digestion of the broken late Cairns intermediate shown in (IV) with BglI or BamHI. VI. Diagram of the predicted outcome of the simple Y shown in panel (V) following neutral 2 d gel electrophoresis and southern blotting. The stall point on the simple Y arc (light green circle) corresponds to the simple Y in panel (V). doi:10.1371/journal.ppat.1003283.g006 the number of incoming viral genomes and with time post- [45]. Moreover, unperturbed viral replication centers display infection in permissive primate cells [29], suggesting that our nascent ssDNA (Sowd, unpublished) and DNA breaks that are ability to detect viral replication centers depends on the ability of likely responsible for activating checkpoint signaling, analogous to each infected cell to generate 10–100 thousand daughter genomes lesions that nucleate host damage response foci. PLOS Pathogens | www.plospathogens.org 10 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure 7. Model of ATM and ATR functions in SV40 DNA replication. (I) Tag initiates viral DNA replication at the viral origin of replication (blue) and the two replication forks progress bidirectionally (red arrowheads). For simplicity, proteins are not shown. (II) Viral DNA replicates quickly until the forks converge to form a late Cairns intermediate (III), which slowly completes replication. (IV) Topoisomerase IIa decatenates fully replicated DNA molecules, yielding two form I daughter molecules. (V) When ATM is inhibited, a one-ended double strand break at a replication fork leads to loss of the replication machinery, while the other fork continues to replicate DNA, generating a rolling circle (VI). (VII) ATM kinase activity facilitates the repair of one-ended double strand breaks. (VIII) When ATR is inhibited, a stalled replication fork remains stable until a functional replication fork approaches it, generating a broken replication intermediate (IX). (X) ATR kinase activity facilitates convergence of moving fork with the stalled fork. We suggest that in the presence of ATM and ATR, repair proteins act on the defective intermediates V and IX to reassemble an intermediate with two functional forks. doi:10.1371/journal.ppat.1003283.g007 PLOS Pathogens | www.plospathogens.org 11 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity A major difference between SV40 replication centers and host might be inaccurately joined with broken host chromatin, damage response foci is that checkpoint signaling does not inhibit contributing to viral tumorigenesis [55]. the viral replication machinery, whereas Chk2 phosphorylation of the purified host replicative helicase Cdc45/Mcm2-7/GINS How does ATR signaling orchestrate SV40 replication fork inhibits its helicase activity in vitro [46] and Chk1 inhibits Cdc45 convergence? recruitment to chromatin to initiate replication in vivo [47]. Based SV40 chromatin replication was highly sensitive to inhibition of on these considerations, we suggest that SV40 replication centers ATR throughout a 48 h infection (Figures 5, S8). One serve as hubs where host replication and repair factors efficiently consequence of ATR inhibition was that infected cells continued service many client viral genomes in close proximity. These hubs to cycle throughout infection, rather than arresting in late S phase are nucleated and maintained by the assembly of the ATM and where viral DNA replication would be favored [30]. However, the ATR signaling complexes at sites of viral replication stress, most prominent SV40 replication defect induced by ATRi was the followed by recruitment of downstream repair factors [1]. Of note, tendency of converging replication forks to stall and break all of the host proteins needed for SV40 DNA replication in vitro (Figures 6, 7, S9). Our data imply that after initiating replication [23,24,25] also function in host DNA repair [23,25,48,49]. Thus at the viral origin, one replisome encounters an unknown SV40, though it encodes only a single essential replication protein, replication block at variable positions in the viral genome has evolved a rather remarkable strategy to generate viral (Figure 6F, S9, I and II, red triangle). Since the two sister Tag replication compartments. helicases need not remain coupled after initiation, they can proceed asynchronously as they replicate the viral genome bidirectionally [26,56,57,58,59]. Thus, the functional, unstalled ATM signaling orchestrates reassembly of viral replisome continues replication until it approaches the stalled fork replication forks, reducing unidirectional replication forks (Figure 6F, III). We suggest that without ATR activity, the Recent studies in several laboratories, including ours, estab- unstalled fork cannot converge with the stalled fork and breaks, lished that knockdown or inhibition of ATM in polyomavirus- yielding the pattern observed on the simple Y arc (Figure 6C, E, F, infected cells reduced production of unit length viral genomes IV–VI). Consistent with this interpretation, fork convergence is [21,22,28,29]. Since these studies evaluated only unit length viral well known to represent a slow step during unperturbed SV40 DNA, the aberrant viral replication products generated by DNA replication in infected cells and to occur in a ,1 kbp region unidirectional replication forks were overlooked (Figures 3, 4, around the BamHI site [60,61,62], suggesting that specialized host S3). Interestingly, total intracellular DNA from unperturbed proteins and ATR-dependent modifications may be needed to infected CV1P cells has also been reported to contain head-to- complete replication. tail SV40 DNA repeats of 50 to 100 kbp at very late times after Our observation that ATRi renders SV40 fork convergence infection [45]. These observations indicate that concatemers may prone to DNA breakage is reminiscent of common fragile sites in be a normal product of viral replication, and suggests that the human genome, which suffer gaps and breaks in Seckel inhibition of ATM activity might simply increase the frequency of Syndrome cells that express defective ATR alleles [63]. Thus unidirectional replication, advance its timing, or both. SV40 and other small DNA tumor virus genomes may harbor a Although replication-associated breaks may be a rare event potential fragile site in the region where the two viral replication during unperturbed viral DNA replication, the large number of forks converge. Consistent with this speculation, C-terminal replicating viral genomes would facilitate their detection, partic- truncation of the polyomaviral T antigen encoded in the ‘‘fragile ularly when ATM activity is suppressed. Yet surprisingly, when site’’ could render an integrated viral genome replication-defective undigested total intracellular DNA from an ATM-inhibited and perhaps more tumorigenic [52,64,65,66]. Similarly, the viral infection was analyzed by 2 d gel electrophoresis, bidirectional ‘‘fragile site’’ where replication forks converge would correspond replication was still observed (data not shown) and unit length viral to common viral genome breakpoints in integrated high risk DNA remained the predominant product when ATM was papillomaviral genomes in cervical cancer [67,68,69]. inhibited (Figures 3 and S3). These observations can be most simply explained by a model in which theta-form SV40 replication Materials and Methods intermediates (Figure 7, I–III) break, giving rise to unidirectional forks that amplify the break by generating concatemers and For details not described below, please refer to the online branched concatemers [38,39] (Figure 7, V, VI). Our data suggest Supporting Methods (Protocol S1). that ATM kinase activity is crucial for the repair of one-ended replication-associated DSBs to reassemble bidirectional replication Use of PIKK inhibitors intermediates (Figure 7, VII) [49,50,51]. Ku-55933, kindly provided by Astra-Zeneca, was used as It is interesting to consider a possible role for unidirectional viral described [12,29]. Importantly, Ku-55933 did not inhibit sixty off- replication and its large concatemeric products in the tumorigenic target kinases. It specifically inhibits purified ATM with an IC50 activity of SV40, and more broadly of polyoma- and papilloma- of 12.9 nM, whereas it inhibits the related kinases mTOR and viruses. Concatemeric genomes of Merkel cell carcinoma virus and DNA-PK with IC values of 2500 nM and 9300 nM, respective- HPV are often integrated into human chromosomal DNA in ly, in vitro [12]. Caffeine (Sigma) was dissolved to 24 mM in tumors associated with these viruses [52,53,54]. The integration DMEM and used at a final concentration of 8 mM to inhibit events and the consequences of long-term viral oncogene ATM and ATR [40]. ATRi and Nu7441 were generous gifts from expression are primary risk factors for such cancers. It seems Dr. David Cortez. ATRi dissolved in DMSO at 5 mM was used at likely that in an infected cell under conditions of insufficient ATM a final concentration of 5 mM [13]. ATRi selectively inhibits ATR activity, the level of viral concatemers would rise. With inadequate with a K of 13 nM, whereas at least a 100-fold higher ATM activity, breaks in host chromosomal DNA would also be concentration is required in vitro to inhibit the related kinases less frequently repaired through accurate, homology-dependent ATM (K = 16000 nM), DNA-PK (K = 2200 nM), mTOR i i repair. Thus one can speculate that viral DNA concatemers (K = 1000 nM), and PI3Kgamma (K = 3900 nM) [13]. Nu7441 i i generated under conditions of insufficient DNA damage signaling was dissolved in DMSO to 2 mM and applied to cells at 1 mM PLOS Pathogens | www.plospathogens.org 12 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity [70,71]. Nu7026 (EMD) was dissolved to 5 mM in DMSO and normalized signal present in the infected solvent control to yield used at a final concentration of 10 mM [72]. the DNA signal (% of DMSO). DMEM containing inhibitor or solvent was added to cells The southern blot signals from an equal area of each arc in 30 min prior to infection. At time zero, DMEM with inhibitor or neutral 2 d gels were quantified (boxed areas in Figure 4C, D, E, solvent was removed, and fresh warm DMEM containing inhibitor F). Background signal in an area of equal size was subtracted, and or solvent and SV40 was added to cells. Cells were gently rocked the values for each arc were normalized to the value for the double every 15 min during the first 2 hpi. At 2 hpi, complete DMEM Y (Figure 4H) or bubble arc (Figure 4I). containing inhibitor or solvent was added to each dish of cells. At 20 hpi, medium was aspirated and cells were washed once with Statistics PBS to remove residual inhibitor or solvent. Fresh medium Statistics were performed in Microsoft Excel using the data containing inhibitor or solvent was then added to cells and analysis package. Prior to t-test, single factor ANOVA analysis was infections were allowed to proceed until the chosen endpoint. performed. If ANOVA resulted in p,0.5, a two sample t-test Solvent control treatments utilized the solvent concentration assuming unequal variances was performed. One-tailed p values present in the inhibitor-treated medium. from student’s t test are denoted by the number of asterisk(s): * p,0.05 ** p,0.01 *** p,0.001 **** p,0.0001. All one tailed p DNA isolation values were generated by comparing data from SV40 infection in Total intracellular DNA was prepared from infected and mock- the presence of inhibitor to that from SV40 infection in the infected cells. For each experiment, all samples were prepared presence of DMSO. Bar graphs present the average of 3 to 4 from an equal number of cells. Cell pellets were resuspended in independent experiments and error bars represent standard 0.4 ml of TE (10 mM Tris pH 8.0, 1 mM EDTA). SDS, RNase deviation. A, proteinase K, and Tris pH 7.5 were added to a final concentration of 0.4%, 0.2 mg/ml, 50 ug/ml and 100 mM, Supporting Information respectively, in a total volume of 0.5 ml. Following overnight Figure S1 Viral replication centers co-localize with host digestion at 37uC, each sample was extracted twice with Tris- DNA replication factors in SV40-infected BSC40 cells. A– saturated phenol (pH 7.9) and once with 24:1 chloroform: isoamyl E. Merged images of chromatin-bound Tag and the indicated host alcohol. DNA was precipitated with sodium acetate and ethanol. DNA replication factors from mock- or SV40-infected BSC40 cells DNA was allowed to dissolve in T0.1E (10 mM Tris pH 8.0, at 48 hpi. Top image for each replication protein is a mock- 0.1 mM EDTA) for 2 days, and then digested overnight at 37uC infected cell. The fluorescence intensity in arbitrary units (AU) with 40 U of SacI-HF and XbaI (both from New England along the line shown in the merged image is graphed in the right Biolabs). Digested DNA was re-precipitated and then dissolved in panel. Scale bars, 10 mm. 50 mL of T0.1E per 2.5610 cells. Equal volumes of DNA were (TIF) loaded on gels for southern blots unless otherwise indicated. Figure S2 Host DNA replication proteins co-localize Agarose gel electrophoresis with Tag in SV40-infected U2OS cells. A–D. Representative One-dimensional 0.7% agarose gels in 16 TAE were electro- images of chromatin-bound Tag and the indicated host DNA phoresed at 10 V/cm for 1.5 h. Neutral 2 d gel electrophoresis replication proteins from SV40-infected U2OS cells at 48 hpi. was performed as previously described [37] with the following The fluorescence intensity in arbitrary units (AU) along the line modifications. The first dimension of the gel was electrophoresed shown in the merged image is graphed in the right panel. Scale at 1 V/cm through a 0.4% 16TAE for 22 h. 16TAE was found bars, 10 mm. to enhance separation of D-loop arc (data not shown). The second (TIF) dimension was electrophoresed at 5.5 V/cm through a 1.1% 16 Figure S3 Aberrant DNA structures accumulate in TBE gel containing 0.5 ng/ml ethidium bromide for 5.5 h with ATM-inhibited SV40-infected U2OS cells. A. Total DNA circulation. extracted at 48 hpi from SV40-infected BSC40 cells treated with Ku-55933 during the indicated phases of infection, as in Figure 2A, Southern blotting analysis was analyzed by southern blot. Lanes 1–5: DNA digested with Southern blotting was performed using radiolabeled probes for XbaI and SacI. Lanes 6–10: DNA digested with BglI. B. Southern SV40 and BSC40 mitochondrial DNA as described [34]. A probe blot of DNA replicated in SV40-infected U2OS cells in the for human mitochondrial DNA was generated by PCR amplifi- presence of ATM inhibitor during the indicated phases of cation (primers: U2OS Mito-F ACG CGA TAG CAT TGC GAG infection. C. Quantification of SV40 signal in monomeric forms AC; U2OS Mito-R CTT TGG GGT TTG GTT GGT TCG), and the whole sample in each lane, normalized to the followed by random priming. Hybridized blots were visualized corresponding signals in the DMSO solvent lane as in panel B. using a Typhoon Trio laser scanning imager (GE Healthcare) and D and E. Fraction of signal in monomer forms (D) or in the quantified using ImageQuant 5.2 (GE Healthcare). indicated DNA structure (E) in DNA extracted at 48 hpi from cells Bands or arcs corresponding to each DNA structure of interest treated with Ku-55933 during the indicated phases of infection as were quantified and the value from a region of the blot without in panel B. Values in C–E represent the average of 3 to 4 signal, e.g. Mock for SV40 probe, was subtracted as background. independent experiments. To compare the level of a DNA structure after a given treatment (EPS) (e.g. DNA structure (% of Total DNA)), the total signals for the Figure S4 Caffeine inhibits ATM and ATR activities in DNA were summed, and the signal of a discrete DNA structure (e.g. form I monomer) were divided by the total signal in the lane SV40-infected BSC40 cells. A. BSC40 cells were treated with caffeine during the indicated phases of a 48 h SV40 infection. B (e.g. [form I monomer signal]/[total signal in the lane]). To quantify variations in replication between treatments, all SV40 and C. Western blots of cell lysates from SV40-infected BSC40 DNA signals were normalized using the respective mitochondrial cells exposed to caffeine as depicted in (A). DNA signal. Normalized signals were then divided by the (TIF) PLOS Pathogens | www.plospathogens.org 13 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity Figure S5 ATM and ATR inhibition increases aberrant from infected U2OS cells treated with ATRi, normalized to the DNA product accumulation. A, E. Southern blots of total corresponding signals from infected cells treated with DMSO. D. DNA extracted from BSC40 (A) or U2OS (E) cells treated with Fraction of total SV40 signal in the indicated DNA structures in caffeine during phases of SV40 infection as described in Figure infected U2OS cells exposed to ATRi. Bars in graphs in C, D S4A. B, F. Quantification of signal in SV40 monomer forms or represent the average of 3 to 4 independent experiments. total DNA in caffeine-treated BSC40 (B) or U2OS (F) cells, (EPS) normalized to that in DMEM solvent. C, D, G, H. SV40 signal in Figure S9 ATR inhibition results in replication fork monomer forms (C and G) or aberrant DNA structures (D and H) stalling and breakage. A. Diagrams of replication intermedi- accumulated in caffeine-treated BSC40 (C and D) or U2OS (G ates on a simple Y arc produced when ATR is inhibited. Cleavage and H) cells, divided by the total SV40 DNA signal in respective sites are denoted as a colored vertical line: BglI (orange), BamHI lane. Bars in B–D and F–H represent the average of 3 to 4 (green). I. Replication begins at the origin and forks diverge independent experiments. bidirectionally to produce theta-form replication intermediates. II. (EPS) Both replisomes progress unless a replication block (red triangle) is Figure S6 DNA-PK activity is dispensable in unper- encountered, causing a fork to stall. III. The stalled replication fork turbed SV40 infection. A. Experimental scheme for treatment is closest to orange BglI site (viral origin of replication). The of BSC40 cells with DNA PK inhibitors during phases of a 48 h functional replisome continues replication and converges with the SV40 infection. B., C. Southern blots of DNA extracted from stalled replication fork. IV. One-sided DSB forms at the BSC40 cells treated as in A with Nu7026 (B) or Nu7441(C). D. replicating fork of the late Cairns intermediate shown in (III) as Quantification of SV40 replication products as in B, C. it approaches the stall site. V. Simple Y DNA structure generated (EPS) by BglI or BamHI digestion of the broken late Cairns intermediate shown in (IV). VI. Diagram of the predicted outcome of the simple Figure S7 ATRi inhibits ATR activity in SV40-infected Y shown in panel (V) after neutral 2 d gel electrophoresis and BSC40 cells. A. Exposure of SV40-infected BSC40 cells to ATRi southern blot analysis. The stall point on the simple Y arc (light during defined phases of a 48 h infection. B. WST-1 viability assay green circle) corresponds to the simple Y in panel (V). of SV40-infected BSC40 cells treated with ATRi as described in A. (EPS) Values were normalized to SV40-infected cells in the presence of DMSO. Error bars represent four independent experiments. C, D. Protocol S1 Supporting methods. Western blot of cell lysates from SV40-infected BSC40 cells (DOCX) exposed to ATRi as indicated. (EPS) Acknowledgments Figure S8 ATR is needed for efficient viral DNA We thank Dr. David Cortez for generously sharing inhibitors and advice, replication in U2OS cells. A. Southern blot analysis of total AstraZeneca for Ku-55933, Dr. E. Kremmer for anti-Cdc45, Dr. DNA from BSC40 cells treated with ATRi during the indicated Katherine Friedman, Dr. Heather Lorimer, and Dr. Bonita Brewer for advice on 2 d gels, the members of the Fanning lab for discussion, and phases of infection as in Figure S7A. Lanes 1–5: DNA digested Ashley Sowd for moral support. with XbaI and SacI. Lanes 6–10: DNA digested with BglI. An equal amount of unit length SV40 DNA was loaded in each lane Author Contributions using the data in figure 5C using an equal number of cells. B. Total DNA from SV40-infected U2OS cells treated with ATRi as Conceived and designed the experiments: GAS EF. Performed the in Figure S7A was analyzed by southern blotting. C. Quantifica- experiments: GAS NYL. Analyzed the data: GAS NYL EF. Wrote the tion of SV40 signal in total and monomeric SV40 DNA forms paper: GAS EF. References 1. Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play 13. Reaper PM, Griffiths MR, Long JM, Charrier JD, Maccormick S, et al. (2011) with knives. Mol Cell 40: 179–204. Selective killing of ATM- or p53-deficient cancer cells through inhibition of 2. Chu WK, Hickson ID (2009) RecQ helicases: multifunctional genome ATR. Nat Chem Biol 7: 428–430. caretakers. Nat Rev Cancer 9: 644–654. 14. Weitzman MD, Lilley CE, Chaurushiya MS (2010) Genomes in conflict: maintaining genome integrity during virus infection. Annu Rev Microbiol 64: 3. Stracker TH, Petrini JH (2011) The MRE11 complex: starting from the ends. 61–81. Nat Rev Mol Cell Biol 12: 90–103. 15. Weitzman MD, Lilley CE, Chaurushiya MS (2011) Changing the ubiquitin 4. Meek K, Dang V, Lees-Miller SP (2008) DNA-PK: the means to justify the ends? landscape during viral manipulation of the DNA damage response. FEBS Lett Adv Immunol 99: 33–58. 585: 2897–2906. 5. Guo Z, Kozlov S, Lavin MF, Person MD, Paull TT (2010) ATM activation by 16. Weller SK (2010) Herpes simplex virus reorganizes the cellular DNA repair and oxidative stress. Science 330: 517–521. protein quality control machinery. PLoS Pathog 6: e1001105. 6. Bakkenist CJ, Kastan MB (2003) DNA damage activates ATM through 17. Lau A, Swinbank KM, Ahmed PS, Taylor DL, Jackson SP, et al. (2005) intermolecular autophosphorylation and dimer dissociation. Nature 421: 499– Suppression of HIV-1 infection by a small molecule inhibitor of the ATM kinase. Nat Cell Biol 7: 493–500. 7. Zou L, Elledge SJ (2003) Sensing DNA damage through ATRIP recognition of 18. Moody CA, Laimins LA (2009) Human papillomaviruses activate the ATM RPA-ssDNA complexes. Science 300: 1542–1548. DNA damage pathway for viral genome amplification upon differentiation. 8. Cimprich KA, Cortez D (2008) ATR: an essential regulator of genome integrity. PLoS Pathog 5: e1000605. Nat Rev Mol Cell Biol 9: 616–627. 19. Sakakibara N, Mitra R, McBride AA (2011) The papillomavirus E1 helicase 9. Matsuoka S, Ballif BA, Smogorzewska A, McDonald ER, 3rd, Hurov KE, et al. activates a cellular DNA damage response in viral replication foci. J Virol 85: (2007) ATM and ATR substrate analysis reveals extensive protein networks 8981–8995. responsive to DNA damage. Science 316: 1160–1166. 20. Wallace NA, Robinson K, Howie HL, Galloway DA (2012) HPV 5 and 8 E6 10. Brown EJ, Baltimore D (2000) ATR disruption leads to chromosomal abrogate ATR activity resulting in increased persistence of UVB induced DNA fragmentation and early embryonic lethality. Genes Dev 14: 397–402. damage. PLoS Pathog 8: e1002807. 11. Casper AM, Nghiem P, Arlt MF, Glover TW (2002) ATR regulates fragile site 21. Dahl J, You J, Benjamin TL (2005) Induction and utilization of an ATM stability. Cell 111: 779–789. signaling pathway by polyomavirus. J Virol 79: 13007–13017. 12. Hickson I, Zhao Y, Richardson CJ, Green SJ, Martin NM, et al. (2004) 22. Jiang M, Zhao L, Gamez M, Imperiale MJ (2012) Roles of ATM and ATR- Identification and characterization of a novel and specific inhibitor of the ataxia- Mediated DNA Damage Responses during Lytic BK Polyomavirus Infection. telangiectasia mutated kinase ATM. Cancer Res 64: 9152–9159. PLoS Pathog 8: e1002898. PLOS Pathogens | www.plospathogens.org 14 April 2013 | Volume 9 | Issue 4 | e1003283 SV40 Replication Fork Integrity 23. Bullock PA (1997) The initiation of simian virus 40 DNA replication in vitro. 49. Hashimoto Y, Puddu F, Costanzo V (2012) RAD51- and MRE11-dependent Crit Rev Biochem Mol Biol 32: 503–568. reassembly of uncoupled CMG helicase complex at collapsed replication forks. 24. Borowiec JA, Dean FB, Bullock PA, Hurwitz J (1990) Binding and unwinding– Nat Struct Mol Biol 19: 17–24. how T antigen engages the SV40 origin of DNA replication. Cell 60: 181–184. 50. Petermann E, Helleday T (2010) Pathways of mammalian replication fork 25. Waga S, Stillman B (1994) Anatomy of a DNA replication fork revealed by restart. Nat Rev Mol Cell Biol 11: 683–687. reconstitution of SV40 DNA replication in vitro. Nature 369: 207–212. 51. Munoz-Galvan S, Tous C, Blanco MG, Schwartz EK, Ehmsen KT, et al. (2012) 26. Sowd GA, Fanning E (2012) A Wolf in Sheep’s Clothing: SV40 Co-opts Host Distinct roles of Mus81, Yen1, Slx1-Slx4, and Rad1 nucleases in the repair of Genome Maintenance Proteins to Replicate Viral DNA. PLoS Pathog 8: replication-born double-strand breaks by sister chromatid exchange. Mol Cell e1002994. Biol 32: 1592–1603. 27. Fanning E, Zhao K (2009) SV40 DNA replication: from the A gene to a 52. Chang Y, Moore PS (2012) Merkel cell carcinoma: a virus-induced human nanomachine. Virology 384: 352–359. cancer. Annu Rev Pathol 7: 123–144. 28. Shi Y, Dodson GE, Shaikh S, Rundell K, Tibbetts RS (2005) Ataxia- 53. DeCaprio JA (2009) Does detection of Merkel cell polyomavirus in Merkel cell telangiectasia-mutated (ATM) is a T-antigen kinase that controls SV40 viral carcinoma provide prognostic information? J Natl Cancer Inst 101: 905–907. replication in vivo. J Biol Chem 280: 40195–40200. 54. DiMaio D, Liao JB (2006) Human papillomaviruses and cervical cancer. Adv 29. Zhao X, Madden-Fuentes RJ, Lou BX, Pipas JM, Gerhardt J, et al. (2008) Virus Res 66: 125–159. Ataxia telangiectasia-mutated damage-signaling kinase- and proteasome-depen- 55. Chia W, Rigby PW (1981) Fate of viral DNA in nonpermissive cells infected with dent destruction of Mre11-Rad50-Nbs1 subunits in Simian virus 40-infected simian virus 40. Proc Natl Acad Sci U S A 78: 6638–6642. primate cells. J Virol 82: 5316–5328. 56. Moarefi IF, Small D, Gilbert I, Hopfner M, Randall SK, et al. (1993) Mutation 30. Rohaly G, Korf K, Dehde S, Dornreiter I (2010) Simian virus 40 activates ATR- of the cyclin-dependent kinase phosphorylation site in simian virus 40 (SV40) Delta p53 signaling to override cell cycle and DNA replication control. J Virol large T antigen specifically blocks SV40 origin DNA unwinding. J Virol 67: 84: 10727–10747. 4992–5002. 31. Tang Q, Bell P, Tegtmeyer P, Maul GG (2000) Replication but not transcription 57. Schneider C, Weisshart K, Guarino LA, Dornreiter I, Fanning E (1994) Species- of simian virus 40 DNA is dependent on nuclear domain 10. J Virol 74: 9694– specific functional interactions of DNA polymerase alpha-primase with simian virus 40 (SV40) T antigen require SV40 origin DNA. Mol Cell Biol 14: 3176– 32. Boichuk S, Hu L, Hein J, Gjoerup OV (2010) Multiple DNA damage signaling and repair pathways deregulated by simian virus 40 large T antigen. J Virol 84: 58. Weisshart K, Taneja P, Jenne A, Herbig U, Simmons DT, et al. (1999) Two 8007–8020. regions of simian virus 40 T antigen determine cooperativity of double-hexamer 33. Hein J, Boichuk S, Wu J, Cheng Y, Freire R, et al. (2009) Simian virus 40 large assembly on the viral origin of DNA replication and promote hexamer T antigen disrupts genome integrity and activates a DNA damage response via interactions during bidirectional origin DNA unwinding. J Virol 73: 2201–2211. Bub1 binding. J Virol 83: 117–127. 59. Yardimci H, Wang X, Loveland AB, Tappin I, Rudner DZ, et al. Bypass of a 34. Zhou B, Arnett DR, Yu X, Brewster A, Sowd GA, et al. (2012) Structural basis protein barrier by a replicative DNA helicase. Nature 492: 205–209. for the interaction of a hexameric replicative helicase with the regulatory subunit 60. Tapper DP, Anderson S, DePamphilis ML (1982) Distribution of replicating of human DNA polymerase alpha-primase. J Biol Chem 287: 26854–26866. simian virus 40 DNA in intact cells and its maturation in isolated nuclei. J Virol 35. Cohen GL, Wright PJ, DeLucia AL, Lewton BA, Anderson ME, et al. (1984) 41: 877–892. Critical spatial requirement within the origin of simian virus 40 DNA 61. Tapper DP, DePamphilis ML (1980) Preferred DNA sites are involved in the replication. J Virol 51: 91–96. arrest and initiation of DNA synthesis during replication of SV40 DNA. Cell 22: 36. Lobrich M, Shibata A, Beucher A, Fisher A, Ensminger M, et al. (2010) 97–108. gammaH2AX foci analysis for monitoring DNA double-strand break repair: 62. Tapper DP, Anderson S, DePamphilis ML (1979) Maturation of replicating strengths, limitations and optimization. Cell Cycle 9: 662–669. simian virus 40 DNA molecules in isolated nuclei by continued bidirectional 37. Friedman KL, Brewer BJ (1995) Analysis of replication intermediates by two- replication to the normal termination region. Biochim Biophys Acta 565: 84–97. dimensional agarose gel electrophoresis. Methods Enzymol 262: 613–627. 63. Casper AM, Durkin SG, Arlt MF, Glover TW (2004) Chromosomal instability 38. Preiser PR, Wilson RJ, Moore PW, McCready S, Hajibagheri MA, et al. (1996) at common fragile sites in Seckel syndrome. Am J Hum Genet 75: 654–660. Recombination associated with replication of malarial mitochondrial DNA. 64. Shuda M, Feng H, Kwun HJ, Rosen ST, Gjoerup O, et al. (2008) T antigen EMBO J 15: 684–693. mutations are a human tumor-specific signature for Merkel cell polyomavirus. 39. Backert S (2002) R-loop-dependent rolling-circle replication and a new model Proc Natl Acad Sci U S A 105: 16272–16277. for DNA concatemer resolution by mitochondrial plasmid mp1. EMBO J 21: 65. Gjoerup O, Chang Y (2010) Update on human polyomaviruses and cancer. Adv 3128–3136. Cancer Res 106: 1–51. 40. Sarkaria JN, Busby EC, Tibbetts RS, Roos P, Taya Y, et al. (1999) Inhibition of 66. An P, Saenz Robles MT, Pipas JM (2012) Large T antigens of polyomaviruses: ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer amazing molecular machines. Annu Rev Microbiol 66: 213–236. Res 59: 4375–4382. 67. Schwarz E, Freese UK, Gissmann L, Mayer W, Roggenbuck B, et al. (1985) 41. Chen BP, Chan DW, Kobayashi J, Burma S, Asaithamby A, et al. (2005) Cell Structure and transcription of human papillomavirus sequences in cervical cycle dependence of DNA-dependent protein kinase phosphorylation in carcinoma cells. Nature 314: 111–114. response to DNA double strand breaks. J Biol Chem 280: 14709–14715. 68. Kadaja M, Isok-Paas H, Laos T, Ustav E, Ustav M (2009) Mechanism of 42. Pohlhaus JR, Kreuzer KN (2006) Formation and processing of stalled replication genomic instability in cells infected with the high-risk human papillomaviruses. forks–utility of two-dimensional agarose gels. Methods Enzymol 409: 477–493. PLoS Pathog 5: e1000397. 43. Lopes M, Cotta-Ramusino C, Pellicioli A, Liberi G, Plevani P, et al. (2001) The 69. Woodman CB, Collins SI, Young LS (2007) The natural history of cervical HPV DNA replication checkpoint response stabilizes stalled replication forks. Nature infection: unresolved issues. Nat Rev Cancer 7: 11–22. 412: 557–561. 70. Hardcastle IR, Cockcroft X, Curtin NJ, El-Murr MD, Leahy JJ, et al. (2005) 44. Lukas J, Lukas C, Bartek J (2011) More than just a focus: The chromatin Discovery of potent chromen-4-one inhibitors of the DNA-dependent protein response to DNA damage and its role in genome integrity maintenance. Nat Cell kinase (DNA-PK) using a small-molecule library approach. J Med Chem 48: Biol 13: 1161–1169. 7829–7846. 45. Rigby PW, Berg P (1978) Does simian virus 40 DNA integrate into cellular DNA 71. Leahy JJ, Golding BT, Griffin RJ, Hardcastle IR, Richardson C, et al. (2004) during productive infection? J Virol 28: 475–489. 46. Ilves I, Tamberg N, Botchan MR (2012) Checkpoint kinase 2 (Chk2) inhibits the Identification of a highly potent and selective DNA-dependent protein kinase activity of the Cdc45/MCM2-7/GINS (CMG) replicative helicase complex. (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Proc Natl Acad Sci U S A 109: 13163–13170. Med Chem Lett 14: 6083–6087. 47. Liu P, Barkley LR, Day T, Bi X, Slater DM, et al. (2006) The Chk1-mediated S- 72. Veuger SJ, Curtin NJ, Richardson CJ, Smith GC, Durkacz BW (2003) phase checkpoint targets initiation factor Cdc45 via a Cdc25A/Cdk2- Radiosensitization and DNA repair inhibition by the combined use of novel independent mechanism. J Biol Chem 281: 30631–30644. inhibitors of DNA-dependent protein kinase and poly(ADP-ribose) polymerase- 48. Lydeard JR, Lipkin-Moore Z, Sheu YJ, Stillman B, Burgers PM, et al. (2010) 1. Cancer Res 63: 6008–6015. Break-induced replication requires all essential DNA replication factors except 73. Hirt B (1967) Selective extraction of polyoma DNA from infected mouse cell those specific for pre-RC assembly. Genes Dev 24: 1133–1144. cultures. J Mol Biol 26: 365–369. PLOS Pathogens | www.plospathogens.org 15 April 2013 | Volume 9 | Issue 4 | e1003283

Journal

PLoS Pathogens – Public Library of Science (PLoS) Journal

Published: Apr 4, 2013

There are no references for this article.