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

Learn More →

Chk1 regulates the density of active replication origins during the vertebrate S phase

Chk1 regulates the density of active replication origins during the vertebrate S phase The EMBO Journal (2007) 26, 2719–2731 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Chk1 regulates the density of active replication origins during the vertebrate S phase 1 2 these sub-chromosomal units is thought to play a direct role Apolinar Maya-Mendoza , Eva Petermann , 3 2 in defining the S-phase programme (Sadoni et al, 2004). David AF Gillespie , Keith W Caldecott 1, In human cells, DNA is duplicated within an S phase of and Dean A Jackson * about 10 h and during this time, roughly 50 000 replication Faculty of Life Sciences, University of Manchester, MIB, Manchester, origins are used to activate synthesis of replicons that are UK, Genome damage and Stability Centre, University of Sussex, Falmer, typically B100 kb in length (Jackson and Pombo, 1998; Brighton, UK and Beatson Institute for Cancer Research, Cancer Research UK, Glasgow, UK Berezney et al, 2000). Potential origins are activated through- out S phase so that B10% of replicons are engaged in DNA The checkpoint kinase 1 (Chk1) preserves genome integ- synthesis at any time. Single-molecule analysis of DNA fibres rity when replication is performed on damaged templates. shows that specific origins or groups of origins function at Recently, Chk1 has also been implicated in regulating specific times of S phase and can be developmentally regu- different aspects of unperturbed S phase. Using mamma- lated (Jackson and Pombo, 1998; Norio et al, 2005). In lian and avian cells with compromised Chk1 activity, we addition, the temporal activation of specific regions of the show that an increase in active replicons compensates for genome correlates with the appearance of distinct patterns of inefficient DNA polymerisation. In the absence of damage, replication sites as cells progress from early to middle and loss of Chk1 activity correlates with the frequent stalling then late S phase (reviewed in Jackson, 1995). and, possibly, collapse of active forks and activation of In somatic vertebrate cells, almost nothing is known about adjacent, previously suppressed, origins. In human cells, the mechanisms that regulate the temporal activation and super-activation of replication origins is restricted to pre- suppression of potential replication origins (Gilbert, 2002; existing replication factories. In avian cells, in contrast, Machida et al, 2005). However, recent studies have suggested Chk1 deletion also correlates with the super-activation of that the replication checkpoint pathways contribute to replication factories and loss of temporal continuity in the the regulation of origin activation during normal S phase replication programme. The same phenotype is induced in (reviewed in Fisher and Mechali, 2004; Kastan and Bartek, wild-type avian cells when Chk1 or ATM/ATR is inhibited. 2004). Although checkpoint pathways are activated in re- These observations show that Chk1 regulates replication sponse to DNA damage (reviewed in Sancar et al, 2004), a origin activation and contributes to S-phase progression in function in unperturbed S phase is implied by the fact that somatic vertebrate cells. checkpoint kinase 1 (Chk1) is essential for normal develop- The EMBO Journal (2007) 26, 2719–2731. doi:10.1038/ ment (Liu et al, 2000; Takai et al, 2000). In the absence of sj.emboj.7601714; Published online 10 May 2007 DNA damage, Chk1 has been shown to regulate the physio- Subject Categories: cell cycle; genome stability & dynamics logical turnover of Cdc25A, which in turn regulates the Keywords: checkpoint proteins; DNA foci; DNA replication; activation of replication origins through the action of CDK2, replication origins; S-phase programme associated A and E type cyclins and Cdc45 (Zhao et al, 2002; Sorensen et al, 2003). This pathway, in principle, provides a mechanism for regulating origin activation throughout S phase (Fisher and Mechali, 2004; Shechter and Gautier, 2005), although how a spatially ordered programme of origin activation might be established remains unclear. Introduction In this report, we have used cells with compromised ATM/ ATR and Chk1 function to explore how the checkpoint path- During cell proliferation, eukaryotic genomes are replicated ways contribute to the local and long-range activation of DNA with high fidelity in order to ensure their genetic integrity. synthesis. We have recently shown that Chk1 is required to Because of their size, the duplication of eukaryotic genomes maintain normal rates of replication fork progression in demands that DNA synthesis is activated at numerous repli- human and avian cells in the absence of DNA damage cation ‘origins’ (Machida et al, 2005). In mammalian cells, (Petermann et al, 2006). In the present study, we have replication origins are activated in small groups (Jackson and analysed how ATM/ATR and Chk1 influence the local activa- Pombo, 1998; Berezney et al, 2000) that are replicated tion of replication origins within replicon clusters and the together within individual replication factories. These repli- spatial organisation of replication foci throughout S phase. con clusters can be visualised as DNA foci and the sequential When Chk1 function is compromised, a 2- to 3-fold increase activation of potential replication origins within groups of in origin density is seen, showing that Chk1 normally func- tions to regulate the density of active origins by suppressing *Corresponding author. Faculty of Life Sciences, University of many of the potential origins that could be activated within Manchester, MIB, 131 Princess Street, Manchester M1 7DN, UK. Tel.: þ 1 0161 306 4255; Fax: þ 1 0161 306 8918; each replicon cluster. In human cells, this super-activation of E-mail: [email protected] replication origins in cells with reduced Chk1 activity is restricted to replicons that are already associated with active Received: 24 October 2006; accepted: 17 April 2007; published replication sites, suggesting that the checkpoint proteins must online: 10 May 2007 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2719 | | Chk1 regulates origin density A Maya-Mendoza et al function in the context of spatial constraints that are defined consistent with Chk1 contributing to the regulation of repli- by the programmed assembly of replication factories. cation origin activation (Miao et al, 2003; Syljuasen et al, 2005). However, formal proof for the super-activation of replication origins was not presented in these studies. Results Using spread DNA fibres (Jackson and Pombo, 1998), we first mapped the structure of active replicons in cells that are Replicon structure Cells with perturbed Chk1 function display a small global deficient for ATR and Chk1 function (Figures 1 and 2). Active increase in DNA synthesis and chromatin-associated Cdc45, replicons were labelled with biotin-dUTP following introduc- A B Figure 1 Chk1 regulates the density of active replication origins in HeLa cells. Replication structures were visualised on spread DNA fibres (A–F) using biotin incorporation (shown in red) to assign replication fork polarity. After spreading and immunolabelling, replicon structure was defined by the distance between (panel A) adjacent origins in replicon clusters and (panel B) sister replication forks. Changes in replicon structure induced by treatment with caffeine (panels C–E) and UCN-01 (panels C, F) were determined. Frequency histograms (panels A–C) show the distribution of separation in kilobase pairs, assigned using spreads like the typical examples shown (panels A, B, D–F). Parameters defining average replicon structure under different conditions are shown (G). To monitor the affect of checkpoint protein inhibitors on replication foci (H), replication sites were pulse labelled for 20 min with BrdU (green) and then for 25 min with IdU (red). Unsynchronised cultures were treated with caffeine or UCN-01 before and throughout first labelling and S-phase cells assigned to early, mid or late S phase. Images are merges of confocal sections, with areas of colocalisation shown in yellow. Scale bars (5mm) are shown on individual images. 2720 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al AB Figure 2 Chk1 regulates replication origin density in diploid human fibroblasts. Replication structures of MRC5 human fibroblasts were visualised using the procedures described in Figure 1. Replicon size (A–D) was measured using the separation of sister forks (panel A) after labelling with biotin-dUTP (panels B–D) and structures in untreated cells compared with those in cells treated with caffeine or UCN-01. BrdU labelling was also used to determine the rate of fork elongation (E–G) under the same conditions. Parameters defining average replicon structure under different conditions are shown (H). The organisation of active replication foci in cells treated with caffeine and UCN-01 was compared with untreated cells (I), as described in Figure 1. Scale bars, 5mm. tion of a very low concentration of precursor into cells. As the labelled tracks allows unambiguous assignment of the daugh- precursor is consumed during the labelling period, immuno- ter forks that emanate from a common, central replication detection of biotin reveals comet-like labelled tracks, where origin (Figure 1A and B). the head of the comet represents the position of replication at As the majority of replicons in vertebrate cells are co- the beginning of the labelling pulse and the tail the point replicated in small replicon clusters, the origin spacing within when the label was consumed. Hence, the structure of these clusters provides a direct measure of the replicon length &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2721 | | Chk1 regulates origin density A Maya-Mendoza et al (Figure 1A). In unsynchronised HeLa cells, adjacent origins synthesis was profoundly altered in the presence of caffeine were, on average, 159 kb apart (Figure 1G), similar to the at the level of individual forks (Figure 1A–G), no defects were value of 145 kb during early S phase (Jackson and Pombo, seen at the level of changes in global sites of DNA synthesis. 1998). Although inter-origin distance measurements are robust, Hence, in the presence of inhibitor, colocalisation of the two this analysis neglects isolated replicons and the outgrowing labels and the maintenance of replication patterns with the forks of replicon clusters, which can be much longer than normal number and structure of replication foci shows that those that are located between the active origins (Berezney inhibitor-induced super-activation of latent origins was re- et al, 2000). To incorporate these forks, we also measured the stricted to replicons that were engaged with active replication separation of the daughter forks of individual replicons factories (Figure 1H). Following the most effective caffeine (Figure 1B). As the cells are unsynchronised, labelled repli- treatment, almost all active centres contained the first and cons are visualised at steady state so that the average second labels (Po0.47 (n ¼ 26) using Student’s t-test to separation of daughter forks at the time of labelling will compare control and caffeine-treated cells). approximate to half the replicon length. In unsynchronised The super-activation of latent origins seen in HeLa cells cells, the expected wide range of daughter fork separations was confirmed in MRC5 cells, which are normal human was seen, with an average of 72 kb (Figure 1B and G), diploid fibroblasts from lung (Figure 2). These diploid fibro- roughly half the average inter-origin spacing. blasts have a more uniform origin spacing and average The distribution of daughter forks was also assessed in separation that is about half that in transformed HeLa cells. unsynchronised cells that had been treated with the kinase Even so, DNA spreads from control and caffeine- or UCN-01- inhibitors caffeine-general inhibitor ATR/ATM kinase activ- treated MRC5 cells (Figure 2A–H) showed a clear 2- to 3-fold ity—and UCN-01—a specific inhibitor of Chk1 autophosphory- increase in origin activation (Figure 2A–D) and a correspond- lation (Figure 1C–G). When caffeine was added either 30 min ing fall in the rate of fork migration (Figure 1E–H) upon before the time of replication labelling (designated 30 caf- inhibition of the ATR/ATM pathways. As for HeLa cells feine) or 30 min before and throughout labelling (Figure 1), the observed super-activation of latent origins in 0 0 (designated 30 –30 caffeine; Figure 1C) a clear approximately MRC5 fibroblasts was restricted to pre-established replication 2- to 3-fold decrease in fork separation was seen (Figure 1G). factories (Figure 2I). Similar results were observed when the Chk1 inhibitor UCN-01 was added 60 min before and throughout labelling, with Caffeine-induced activation of latent replication origins replication structures B2-fold closer than in untreated cells We next used a double labelling strategy to define the (Figure 1C and G); slight differences in the caffeine- and UCN-01- relationship between origins that were induced in response induced responses may imply that ATM/ATR also inhibit fork to replication stress and the previously active forks movement through other mechanisms that are independent (Figure 3). Cells were preincubated in caffeine and pulse of Chk1. In both cases, these increases in origin density are labelled for 20 min with bromo-deoxyuridine (BrdU) in pre- conservative estimates because the analysis monitors paired sence of caffeine, washed and then pulse labelled with biotin- sister forks but does not score conjoined forks from origins dUTP (Figure 3A). This labelling protocol defines five classes that initiated synthesis during the labelling pulse. of replication structure (Figure 3B). For both HeLa and MRC5 A notable feature of this experiment is that the increased (Figure 3C), B67% of forks were labelled consecutively with origin density results in a clear decrease in the proportion of the first and second labels (class 1), as expected for forks that widely spaced forks. While cells must contain the natural were elongating throughout the labelling period. In both cell distribution of forks at the beginning of this experiment, the types, 16% of labelled structures contained two forks grow- relative decrease in remote daughter fork results from two ing from a common origin (class 2) and a similar number of factors: (1) novel initiation events dominate the analysis growing forks fused and so terminated during labelling (class 3). because of the high frequency of caffeine-induced origin In controls, very few tracks were labelled uniquely with the activation. These recently activated origins with close sister first or second label (class 4). forks (Figure 1E and F) represent the major population seen In the presence of caffeine, the rate of fork elongation was in the randomly selected fields analysed and (2) widely reduced by at least two-fold throughout the labelling period separated forks are inevitably from long replicons, with (compare control and caffeine class 1 forks in Figure 3B). In replication complexes engaged throughout the labelling pro- addition, quite different proportions of replicating structures tocol. Reduced efficacy of replication at some forks compro- were seen in caffeine treated samples (Figure 3C). Strikingly, mises the structure and assignment of these forks as bona the proportion of consecutively labelled forks (class 1) fell by fide sister pairs. Even so, if spreads are scanned using biased 43-fold as a result of two factors. First, caffeine treatment selection criteria, the active forks in long replicons can be resulted in a 42-fold increase in the number of new initiation found. events during both the first (class 2) and second (class 4) labelling periods. Second, a dramatic increase in the fre- Global analysis of replication in caffeine-treated cells quency of closely spaced active origins (class 5) was also We next characterised how defects in the ATM/ATR pathways seen. Despite a clear fall in the proportion of elongation forks, influence the global structure of replication sites, in order to o5% of forks (class 4—green only) stalled during the first evaluate if the initiation events induced by caffeine and pulse and remained inactive during the second. These ob- UCN-01 were distributed throughout the nucleus. Changes servations show that in the presence of caffeine, active forks in the structure of the replication programme were assessed frequently stall and sometimes collapse, and that these defects using simple labelling and indirect immunofluorescence in replication are compensated for by the activation of novel, (Supplementary Figure S1), and double labelling caffeine-dependent replication origins. A diagnostic feature of (Figure 1H). This analysis demonstrates that whereas DNA novel origin activation is the dramatic increase in interspersed 2722 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al Figure 3 Activation of latent origins in human cells treated with caffeine. HeLa and MRC5 cells were treated with caffeine and pulse labelled with BrdU and biotin-11-dUTP, as shown (A). DNA fibres were prepared, treated with acid and immunolabelled to visualise BrdU (green) and biotin-dU (red). Five classes of replication structure were defined (B; classes 1–5) and the relative frequency of occurrence of the different classes was scored (frequency histograms (C)). Scale bars, 5mm. labelling (class 5). Figure 3B (class 5 caffeine) shows a typical Detailed analysis of replicon structure in DT40 cells extreme case with a 75 kb region containing four active showed that an increased origin density compensates for origins. Clearly, this origin density is very much higher than the reduced fork rate (Figure 4). Replicons of wild-type (wt) is normally seen in control cells (Figures 1 and 2). DT40 cells were smaller than those of HeLa cells, with In the presence of caffeine, this increased density of active an average daughter fork separation of 36.7 kb and origin forks, together with a high frequency of clustered caffeine- spacing of 67.8 kb (Figure 4). Replicons in Chk1-deficient dependent initiation events in the vicinity of previously active DT40 cells were even smaller than those in wt control cells forks, confirms that the vast majority of novel initiation (Po2.7E-19), with an average daughter fork separation of events occur in already active replicon clusters. Under these 24.2 kb and average origin spacing of 47.3 kb. This represents conditions, the efficacy of replication is severely compro- a 1.5- to 2-fold increase in the number of active origins in mised, with all forks showing reduced rates of elongation, Chk1-deficient DT40 cells, which is sufficient to maintain the consistent with frequent stalling during synthesis. Despite normal S-phase duration when individual forks polymerise this reduced rate, a majority of forks continue synthesis and nucleotides at B50% of the rate in wt cells (Petermann et al, only a minority collapse (Figure 3). Importantly, this experi- 2006). ment shows that active replicons contain latent origins ahead As for HeLa and MRC5 cells, clear reductions in daughter of the growing fork, which are suppressed under normal fork separation were seen in DT40 cells treated with UCN-01 conditions but activated in response to replicative stress. (Po2.52E-12) or caffeine (Po1.19E-21) (Figure 4B and D); similar reductions in fork to fork distance were seen follow- Organisation of DNA replication in Chk1 cells ing treatment with caffeine and UCN-01 (Po0.43). A very Because inhibitors such as caffeine might alter or stress the similar replicon structure was also seen in Chk1 DT40 normal pathways that regulate the activation of replication cells (Po0.548 Chk1 versus wt DT40 incubated with origins, we next wanted to analyse if DNA synthesis was caffeine; Figure 4C and D). This shows that DT40 cells have perturbed in Chk1-deficient cells. For this purpose, we chose a very similar density of active replication origins when either to use Chk1-deficient DT40 (Zachos et al, 2003), as Chk-1 ATR/ATM or Chk1 function is compromised and implies that depletion in mammalian model systems is known to be cell Chk1 plays a major role in regulating active origin density in lethal (Liu et al, 2000). Previously, we showed that Chk1- these cells. deficient DT40 cells have a reduced rate of replication at individual forks (Petermann et al, 2006), which appears The replication programme in DT40 cells contrary to the observed increase in global DNA synthesis Defects in origin activation in Chk1-deficient DT40 cells when UCN-01 was used to disrupt Chk1 function in mamma- prompted us to evaluate how the absence of Chk1 influenced lian cells (Syljuasen et al, 2005). the S-phase programme in these cells. As for HeLa cells &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2723 | | Chk1 regulates origin density A Maya-Mendoza et al A BC DT40 chk1–/– D DT40 chk1–/– cells E G DT40 Chk1–/– DT40 Chk2–/– Figure 4 Inhibition of ATR or Chk1 mimics the replication phenotype of Chk1-deleted DT40. Active replicons in Chk1 and isogenically matched wt DT40 cells were analysed after pulse labelling with biotin-11-dUTP. Replication structures were visualised on spread DNA fibres as in Figure 1. Changes in replicon structure in cells with compromised Chk1 activity are shown (frequency histograms (A–C)). In wt DT40, caffeine and UCN-01 (panel B) produced a significant decrease in replicon size (Po1.197E-21 and 2.52E-12, respectively, versus controls). No difference was seen when Chk1 DT40 cultures (panel C) were treated with caffeine (P¼ 0.541). Parameters defining average replicon structure under different conditions are shown (D). The S-phase programme of wt DT40 was also compared with Chk1- and Chk2-deficient cells. Cells were pulse labelled for 20 min with BrdU, immunolabelled (E) and the proportion (F) of early-, mid- and late-S-phase patterns determined. Patterns that did not conform to the recognised structural and spatial criteria were designated as mixed. Caffeine-induced changes in the organisation of replication foci (G) were assessed by double labelling, as described in Figure 1H. High-power details are from the boxed areas shown. Scale bars (5 and 0.5mm in Detail) are shown on individual images. (Figure 1), the S-phase programme of wt DT40 cells (Zachos and Supplementary Figure S2). Chk2-deficient DT40 cells, in et al, 2003) can be described by the size, complexity and contrast, displayed the normal distribution of replication nuclear distribution of their replication foci (Figure 4E–G and patterns, with no significant changes in the structure or Supplementary Figure S2). Using wt DT40, patterns that are number of replication foci (Figure 4E and Supplementary characteristic for this cell type were seen using different Figure S2). These observations imply that the natural link labelling protocols, which allow multiple labelling without between DNA synthesis and the spatial organisation of active reagent cross-talk in fixed or living cells (Figure 4E and replication foci is compromised in Chk1-deficient DT40 cells. Supplementary Figure S2). Chk1-deficient cells had the same range of replication structures as parental DT40 con- Defects in the DT40 replication programme trols. However, it was immediately obvious that the complex- Quantitative analysis of early-, mid- and late-S-phase patterns ity of foci in these cells was greater than in controls. In early showed that Chk1-deficient cells had a higher proportion of S phase, high-resolution 3D analysis of replication foci showed mixed S-phase structures than wt controls (Figure 4F). To DT40 cells to have 3907176 (n ¼ 10) foci, whereas Chk1- extend this observation, we used double labelling protocols deficient cells, with 7557425 (n ¼ 7) foci, had roughly two- to describe the spatial architecture of the S-phase programme fold more active sites. The replication patterns in Chk1 (Figure 4G). The vertebrate S phase is structured so that the and wt cells were shown to be statistically different using the sequential activation of replicon clusters occurs at spatially Mann–Whitney test (Po0.02; U-value 59). Notably, the foci adjacent sites as S phase proceeds (Sadoni et al, 2004). This in Chk1-deficient cells were smaller and more homogeneous spatial relationship is maintained in wt DT40 (Figure 4G), in structure, independently of labelling intensity (Figure 4E where 8177% of foci showed colocalisation when the two 2724 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al pulse labels were applied consecutively. Notably, when dou- of B250 kb, with two adjacent active replicons, were ana- ble labelling was performed in Chk1-deficient cells, colocali- lysed. Typical fibres from DT40 cells displayed four active sation was only seen at 59716% of foci (Figure 4G, Detail forks that were each labelled throughout both the first and and Supplementary Figure S2D), which was very similar to second pulses (Figure 5C). The two origins in this cluster are the colocalisation in both wt and Chk1 DT40 treated with 32.84 mm (85 kb) apart and all four of the active forks show caffeine (Supplementary Figure S2D). Hence, Chk1-deficient similar rates of elongation (Figure 5C). Figure 5D shows a cells show a 42-fold increase in the number of active foci similarly labelled DNA fibre from Chk1-deficient cells, in that are spatially uncoupled from foci that were labelled which an additional origin (þ ) was activated between the immediately upstream in the replication programme. From two already active origins, during the second pulse label. these experiments, we conclude that Chk1 contributes to the Such initiation within B10 kb of two converging forks is mechanism that normally regulates S-phase progression in extremely uncommon in wt cells, but not unusual, if Chk1 DT40 cells. function is perturbed (e.g. Figures 1F and 5A) and is con- Following treatment with caffeine, the structure of replica- sistent with a role of Chk1 in regulating the activation of tion foci in wt DT40 cells mimicked those seen in Chk1- potential replication origins during the normal S phase. deficient cells (Figure 4G). After 50 min in the presence of caffeine, a B2-fold increase in the complexity of early- S-phase foci was seen, although no increase was seen when Aberrant initiation events in Chk1-deficient cells are Chk1-deficient cells were treated with caffeine (Figure 4G and linked to defects in active replication forks Supplementary Figure S3). Similar observations were re- We next explored if aberrant initiation events in cells with corded in cells treated with UCN-01 (Supplementary Figure compromised Chk1 activity were related to intrinsic defects S3). As for Chk1 DT40, double labelling of replication foci in DNA replication (Syljuasen et al, 2005; Petermann et al, in DT40 cells treated with Chk1 inhibitors showed a B2-fold 2006). Normally, BrdU in DNA is only detected by immuno- increase in foci that were not colocalised during consecutive labelling if DNA is first denatured (typically by HCl treat- pulse labels (Figure 4G, Detail and Supplementary Figure S2). ment). However, BrdU incorporated into the DNA of Chk1- This is consistent with disorganisation of the replication deficient cells is readily detectable without denaturation and programme in the presence of Chk1 inhibitors and shows TUNEL analysis confirms that this is due to persistent DNA that caffeine and UCN-01 phenocopy Chk1 deletion in DT40 nicks (Syljuasen et al, 2005; Petermann et al, 2006). cells. Cells were pulse labelled consecutively with BrdU and biotin-dUTP and labelled tracks of spread DNA fibres visua- Unprogrammed initiation of DNA synthesis lised either without or with prior acid treatment (Figure 6 and in Chk1 DT40 Supplementary Figure S5). When immunolabelling was per- To look more precisely at the specific roles of Chk1, we also formed after acid treatment, 490% of forks had adjacent analysed if the activation of DNA synthesis was perturbed in tracks labelled with BrdU and biotin-dU, in both wt and Chk1-deficient DT40 cells under normal—unstressed— Chk1-deficient cells (Figure 6; (þ )HCl). However, when growth conditions (Figure 5). Cells were labelled for 24 h acid treatment was omitted, incorporated BrdU was almost with BrdU before pulse labelling with biotin-dUTP. After completely undetected in spreads from wt cells, with o2% of spreading, both analogues were visualised by indirect immuno- biotin tracks showing very weak (background) BrdU immu- labelling and the distribution of biotin-labelled replication nostaining (Figure 6A, ()HCl). In contrast, 16% of biotin forks was analysed in single, unbroken DNA fibres of tracks in spreads from Chk1-deficient cells had adjacent B250 kb. In a typical experiment (Figure 5A and B), labelled regions of BrdU immunostaining (Figure 6B, ()HCl and regions of isolated (single), unbroken DNA strands were Supplementary Figure S5). These labelled regions were typi- selected at random (using BrdU labelling) and aberrant cally 2.5–10 kb long and the fragmented labelling was clearly replication structures within 25 kb of the paired forks of different in structure to the typical B25 kb tracks seen after replicon clusters with two active replicons were recorded HCl treatment. Notably, the acid-independent BrdU labelling (Figure 5A). In wt DT40, 93.5% of labelled replicons con- in Chk1-deficient cells was frequently adjacent to a newly formed to the expected structure, with pairs of divergent activated origin. A typical example is shown (Figure 6B, sister forks. Only 6.5% had any associated unexpected label- ()HCl and bottom diagram in 6C), where fragmented ling (n ¼ 46; average length of labelled region 93.9 kb). In BrdU labelled overB10 kb lies adjacent to a pair of divergent, contrast, 35% of randomly selected replication structures biotin-labelled forks. Clearly, these biotin-labelled forks from Chk1-deficient DT40 (Figure 5A and B) had associated resulted from the activation of a novel replication origin aberrant replication (n ¼ 40; average length of labelled region towards the end of the BrdU labelling period, at a position 56.5 kb). that is B12 kb from the boundary of the BrdU labelling. As This B5-fold increase of aberrant replication structures in the DNA adjacent to the BrdU boundary is labelled with Chk1-deficient cells is consistent with the activation of novel biotin, the boundary region must represent a termination site, origins, which normally would only occur under conditions where the biotin-labelled and stalled or aberrant BrdU- of replicative stress (Woodward et al, 2006). To emphasise labelled forks met (Figure 5C). this point, we developed a triple labelling protocol, which This experiment makes two key points: (a) that replication provides a more precise description of active replicon struc- forks in Chk1-deficient cells are intrinsically unstable and ture (Figure 5). Cells were labelled for 24 h with BrdU and prone to accumulate ssDNA breaks over many kilobase pulse labelled sequentially with digoxigenin-dUTP and biotin- pairs and (b) that at some forks this damage correlates dUTP, and spread DNA fibres were immunolabelled (Figure 5C with the activation of latent origins that typically are located and D and Supplementary Figure S4). Undamaged DNA fibres 10–20 kb away. &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2725 | | Chk1 regulates origin density A Maya-Mendoza et al Structure of replicons and DNA foci in chick embryonic cells were treated with inhibitors of the checkpoint pathways fibroblasts as describe before. As for human and DT40 cells, CEFs grown As checkpoint pathways may be perturbed in cells, such as in caffeine or UCN-01 showed a B2-fold increase in replica- DT40, that are adapted to long-term culture, we next con- tion fork density, consistent with increased origin activation firmed the organisation of replicons and DNA foci in primary when Chk1 function was inhibited (Figure 7A and C). This avian cells. Early-passage chick embryonic fibroblasts (CEF) increased origin density occurred in cells with B50% of the DT40 wt n=46 DT40 Chk–/– n=46 2nd 1st pulse 2nd 2nd 1st pulse 2nd 2726 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al natural rate of fork elongation (Figure 7B and C). As with Discussion DT40 cells (Figure 4), in the presence of caffeine and UCN-01, CEFs showed a significant (B2-fold) increase in the number Under conditions of replicative stress, eukaryotic cells ex- of their active replication foci (Figure 7D and E and press sophisticated checkpoint pathways that serve to ensure Supplementary Figure S2), which were spatially disorganised the preservation of genomic integrity. If DNA is damaged relative to untreated cells (Figure 7F). during S phase, activation of the intra-S-phase checkpoint A B (–) HCI (–) HCI Merge Merge (–) HCI (–) HCI Merge Merge (–) HCI Biotin (–) HCI BrdU (+) HCI Merge (+) HCI Merge (+) HCI (+) HCI (–) HCI (+) HCI (–) HCI Figure 6 Novel origin activation is spatially coupled to aberrant replication structures in Chk1 DT40 cells. wt and Chk1-deficient DT40 cells were pulse labelled consecutively with BrdU (20 min) and biotin-dUTP (30 min). Spread DNA fibres were immunolabelled to visualise sites of incorporation of BrdU (green) and biotin-dU (red). Labelling was performed either with or without prior treatment with HCl, as shown (A, B). Merged images show overlays of green and red channels (panels A, B). In panel B, the merged image and individual channels are shown for a high-power view of a single fibre. A diagram describing the analysis is shown (C), using the nomenclature described in Figure 5. X indicates a boundary between regions labelled in the first pulse and a nearby replicon that was labelled predominantly during the second, as seen in panel B (high-power example). Scale bars (5 or 10mm) are shown on individual images. Figure 5 Unprogrammed activation of DNA synthesis in Chk1-deficient DT40 cells. Chk1 and isogenically matched wt DT40 cells were labelled with BrdU for 24 h and pulse labelled with biotin-dUTP (30 min; (A, B)) or Dig-dUTP, followed by biotin-11-dUTP (both 30 min; (C, D)). In panel (A) are shown sites of biotin-dU incorporation (red) in single, isolated DNA fibres from wt and Chk1 DT40 cells. DNA fibres were labelled with BrdU to ensure that only single fibres were used for the analysis; for simplicity the BrdU labelling is not shown (some examples are provided in Supplementary Figure S4 and S5). Replicon structure was defined by the organisation of the growing forks (diagram (panel A); filled triangles represent the direction of fork growth from central origins, depicted by open arrows; þ represents a novel or aberrant replication structure) using fibres with two active replicons (four labelled forks). The relative frequency of normal (bidirectional) and aberrant replication structures (within 25 kb of normal forks) was scored (panel B). Unprogrammed initiation in Chk1 cells was confirmed using a combination of Dig-dU and biotin-dU to define replicon structure (panels C, D). DNA fibres were immunolabelled to visualise isolated fibres with two active origins (B250 kb DNA). Such replicons contain two pairs of growing forks that were labelled first with Dig and then with biotin (panels C, D). In Chk1 DT40 (panel D), but not in wt controls (panel C), unprogrammed initiation contributes to synthesis of the unreplicated DNA between the active growing forks—in this example, a new initiation event ( þ ) occurred in the centre of the fibre during the second pulse. Diagrams of the structure of the labelled replicons are shown (panels C, D), using nomenclature described in panel A. Scale bars (5mm (panel A) and 10mm (panels C, D)). &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2727 | | Chk1 regulates origin density A Maya-Mendoza et al AB 30′ caffeine 30′–30′ caffeine 30′–20′ caffeine 5 mM 1 h–30′ UCN-01 30′–20′ caffeine 10 mM 30′ caffeine 5 mM fork to fork 30′–30′ caffeine 5 mM fork to fork 1 h–30′ 300 nM UCN-01 fork to fork 30′–20′ caffeine 5 mM. Fork length BrdU 30′–20′ caffeine 10 mM. Fork length BrdU 30′ caffeine 30′–30′ caffeine UCN-01 30′ caffeine 30′–30′ caffeine Figure 7 Chk1 regulates replication origin density in primary avian fibroblasts. Replication structures of chick embryo fibroblasts were visualised using the procedures described in Figure 2. Replicon size (A–C) was measured using the separation of sister forks (panel A) after labelling with biotin-dU and structures in untreated cells compared with those in cells treated with caffeine or UCN-01. BrdU labelling was also used to determine the rate of fork elongation (panel B) under the same conditions. Parameters defining average replicon structure under different conditions are shown (panel C). The number of active replication factories (D, E) and organisation of active replication foci (F) in cells treated with caffeine and UCN-01 were compared with untreated cells. Changes in the number of active DNA foci in treated cells were shown to 0 0 0 be statistically significant using Mann–Whitney test: 30 caffeine versus control (Po0.05; U-value 78); 30 –30 caffeine versus control (Po0.005; U-value 88); UCN-01 versus control (Po0.02; U-value 82). Scale bars (5 and 0.5mm in Detail) are shown on individual images. maintains replication fork stability and blocks initiation of (Miao et al, 2003; Syljuasen et al, 2005), consistent with Chk1 latent replication origins (Kastan and Bartek, 2004; Sancar contributing to the regulation of DNA synthesis. In support of et al, 2004). During the damage response, activation of Chk1 this idea, recent studies from embryonic systems have sug- leads to phosphorylation of Cdc25, which prevents down- gested that the ATR/Chk1-dependent regulation of origin stream activation of the CDK/cyclin complexes that are function might define the sequential activation of replication required to activate latent origins. By maintaining the phy- origins during S phase (discussed in Fisher and Mechali, siological balance of this pathway, Chk1 is also thought to 2004; Shechter and Gautier, 2005). However, a global increase contribute to the regulation of DNA synthesis in the absence in DNA synthesis in cells with reduced Chk1 activity conflicts of replicative stress (Zhao et al, 2002; Sorensen et al, 2003). with our recent observation that loss of Chk1 function The importance of balance within this regulatory pathway is reduces the rate of replication fork progression to B50% of þ / emphasised by the phenotype of Chk1 cells from trans- that in wt cells (Petermann et al, 2006). In order to resolve genic mice (Lam et al, 2004). Such cells maintain three clear this conflict, we have used quantitative analysis of nascent haploinsufficiency phenotypes: inappropriate S-phase entry, replicons in spread DNA fibres. Using both transformed and accumulation of DNA damage during replication and failure primary cells of human and avian origin, this approach to restrain mitotic entry. Inappropriate cell cycle transitions in provides the first formal proof that the density of active these cells correlate with maintenance of high levels of replication origins is increased by at least 2- to 3-fold when Cdc25A. Chk1 activity is compromised (Figures 1, 2, 4 and 7). In somatic mammalian cells, loss of Chk1 function corre- Consistent with a role for Chk1 in modulating the density lates with a slight (20–30%) increase in global replication of active replication origins during normal S phase, our 2728 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al analysis of DNA fibres showed that inhibition of Chk1 ride Chk1-dependent suppression of late replicons in an compromises the efficacy of DNA replication and leads to early-S-phase cell (Shechter et al, 2004), the late origins of reduced rates of elongation (Figures 2 and 7). Compromised somatic human cells are not activated because they are replication at stalling forks correlates with an accumulation unable to access active replication machinery. This implies of aberrant DNA structures and activation of secondary, that the temporal programme of replication factory assembly previously latent, origins (Figures 3 and 6) within the vicinity plays a dominant role in defining which potential origins can of the damaged forks. This activation of normally suppressed be activated at different times of S phase. origins in cells with compromised Chk1 activity accounts for In avian cells in contrast, loss of Chk1 function correlated the observed increase in density of active origins, and implies with a significant increase in the number of active replication that somatic vertebrate cells have an innate mechanism that foci and some disorganisation of the temporal programme of balances the rate of elongation at replication forks with the S-phase synthesis (Figures 4 and 7). This difference between density of active replication origins. human and avian cells raises interesting questions concern- Alterations in the balance of the normal regulatory ma- ing the evolution of S-phase regulation. ATR and Chk1 play a chinery appear to account—at least in part—for our observa- key role in local (within individual replication factories) tions. It is known that reduced expression or inhibition of regulation of origin activation in early stages of amphibian Chk1 leads to accumulation of Cdc25A, which in principle (Xenopus) development and in avian and mammalian so- would support super-activation of replication origins matic cells. However, in the in vitro Xenopus system, (Sorensen et al, 2003; Lam et al, 2004). We confirm that the temporal activation of replicon clusters is lost if ATR is Cdc25A concentration increases by B5-fold in wt DT40 cell inhibited (Marheineke and Hyrien, 2004). In human cells, treated with caffeine or UCN-01 (Supplementary Figure S3), in contrast, S-phase timing is preserved when ATR function and that the origin associated protein Cdc45 is increased by is compromised (Figures 1 and 2). Intriguingly, somatic about two-fold in human fibroblasts (Supplementary Figure S1) avian cells seem to have evolved a hybrid organisation and DT40 (Supplementary Figure S3) when Chk1 activity is in which the timing programme is disordered (Figures 4 inhibited. and 7) but the duration of S phase is maintained (Zachos et al, 2005). These differences presumably imply that the Does Chk1 contribute to S-phase progression in somatic regulatory pathways that control S-phase progression are able vertebrate cells? to interact (i.e. complement) to different extents in mamma- lian and avian cells. This conclusion is not entirely un- In nuclei assembled using Xenopus egg extract, ATR/Chk1 founded, as deletion of Chk1 in the mouse is known to be regulate the sequential activation of early and late replication cell lethal (Liu et al, 2000) whereas avian DT40 Chk1 cells origins (Marheineke and Hyrien, 2004; Shechter et al, 2004). are viable. It is important to recognise, however, that this embryonic system is very different from somatic cells in key respects. First, assembled nuclei have simple chromatin architecture and no transcription and second, the cell cycle is very rapid, A model for Chk1 function during normal S phase with an S phase of B30 min and correspondingly short Experiments present here support the idea that Chk1 con- replicons of B15 kb (Blow et al, 2001). Although replicon tributes to the efficacy of replication at active replication clusters are activated throughout this short S phase (Blow forks and couples defects in synthesis at active forks to the et al, 2001), it is unknown if this is of physiological signifi- local activation of nearby latent origins. This implies that cance or reflects the stochastic nature of replication in this even during a normal S phase, a Chk1-dependent system system. In clear contrast, somatic mammalian cells have been continually senses the status of active replicons within repli- shown to have well-established S-phase programmes, with cation factories and monitors the number of active forks to specific regions of the genome replicated at precise times ensure progress of the synthetic process. (Jackson and Pombo, 1998; Sadoni et al, 2004). Progress An important feature of this model is the provision of through the programme can be correlated with the spatial latent origins, which are suppressed during normal synthesis architecture of the active replication sites (Jackson, 1995) and but serve to rescue replication if for any reason the active concomitantly correlated with the assembly of different forks might fail. In fact, it has been known for many years classes of chromatin at different times during S phase that immediately before S phase, replicons have B10-fold (Zhang et al, 2002). excess of potential replication origins—defined by MCM2–7 Despite these clear differences, it is interesting to compare complexes—which are clustered into licensing groups (dis- our studies with in vitro observations in Xenopus extracts cussed in Woodward et al, 2006). The redundant MCM (Marheineke and Hyrien, 2004; Shechter et al, 2004), which complexes are displaced from chromatin during replication, suggested that ATR and Chk1 regulate S-phase progression by but also appear to provide a rescue function if active forks are suppressing the activation of late origins during early S phase aborted because of DNA damage (Woodward et al, 2006). (Shechter et al, 2004). In human cells, we show that a This means that under normal conditions, only a minority of mechanism that involves Chk1 suppresses the majority of potential origins is actually used, and that a mechanism must potential origins that exist in active replicons; potential operate to limit origin activation so that once the required origins are known to outnumber those that are used by at number of origins has been activated the use of proximal least 10-fold (discussed in Woodward et al, 2006). When potential origins is suppressed. The data presented here Chk1 activity was inhibited, super-activation of latent origins support the idea (Shechter and Gautier, 2005) that the ATR/ was restricted to regions of the genome that were already Chk1 pathway controls the activation and suppression of engaged with active replication factories (Figures 1H and 2I). replication origins to regulate the density of active origins Hence, while caffeine treatment might be expected to over- throughout S phase of somatic vertebrate cells. &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2729 | | Chk1 regulates origin density A Maya-Mendoza et al 10 min at 01C. Coverslips were then rinsed in PBS and incubated in Materials and methods fresh medium for 30 min. MRC5 and CEF cells were transfected directly with 30ml of transfection mix in 35 mm Petri dishes and Cell culture incubated on ice for 10 min, rinsed with cold PBS and incubated in HeLa cells were grown in DMEM (Sigma) with 5% FBS and fresh medium for 30 min. antibiotics. DT40 cells were grown in RPMI-1640 (Sigma) with 10% These loading conditions use very low precursor concentrations FBS, 1% chicken serum, 10 M b-mercaptoethanol and antibiotics. that were optimised to transiently support normal rates of fork Early-passage MRC5 cells (ATCC cell line CCL-171—normal human elongation. The treatment has no short- or long-term effects on diploid fibroblasts from lung) were grown in MEM, L-glutamine, cell cycle duration and cell viability. For example, replication sodium pyruvate, non-essential amino acids with 10% FBS forks in DT40 cells analysed 10 min after loading travelled and antibiotics. CEF (Kilbey et al, 1996) were grown in DMEM, 12.1972.61 kb (n¼ 52), which corresponds with the average L-glutamine, non-essential amino acids, sodium pyruvate, 1% of elongation rate of 1.2 kb/min in these cells (Petermann et al, chicken serum, 10% FBS and antibiotics. 2006). In addition, the Chk1-dependent DNA damage checkpoint was not activated by these loading and replication conditions DNA fibre experiments (Supplementary Figure S4). Replication tracks were labelled in culture medium containing 25mM BrdU. For dual labelling, cultures were pulse labelled with Immunofluorescence and direct labelling of DNA foci 25mM BrdU, washed and then labelled with 250mM iodo- / / 5 wt, Chk1 and Chk2 DT40 cells at 510 cells/ml were deoxyuridine (IdU). These conditions are known to support natural labelled with 25mM BrdU, 250mM IdU or transfected with the rates of fork elongation (Petermann et al, 2006). Cells were labelled specific dUTP analogue. Cells were washed 3 in cold PBS and with modified dUTPs as detailed below. UCN-01 was used at 300 nm 110 cells swollen in 0.075 M KCl for 15 min at 371C (Jackson and and caffeine at 5 mM. Pombo, 1998). Nuclei were fixed with methanol/acetic acid (3:1), DNA fibre spreads were prepared as previously described added dropwise onto washed microscope slides, and dried. HCl (Jackson and Pombo, 1998). BrdU-labelled tracks were detected denaturation and immunolabelling were performed as detailed with BrdU anti-sheep antibody (Biodesign; M20105S; 1:1000 before (Jackson and Pombo, 1998). dilution; 1 h at 201C) using either Cy3- or AlexaFluor-488- For double labelling experiments, DT40 nuclei fixed with conjugated donkey anti-sheep secondary antibody. Biotin-11-dUTP methanol/acetic acid or HeLa, MRC5 and CEF cells fixed in 4% detection was carried out using a mouse monoclonal antibody PF were rinsed three times in PBS, two times in ddH O and then (Clone BN-34; Sigma; 1:1000 dilution; 1 h at 201C) and Dig-11-dUTP 2 incubated with HCl. The slides were rinsed in PBS and PBSþ , (Roche) detection using a sheep polyclonal antibody (Roche; 1:1000 blocked and incubated with rat anti-BrdU (Immunologicals Direct dilution; 0.5 h at 201C) and appropriate Cy3- or AlexaFluor-488- Clone BU 1/75; 1:1000 dilution; 1 h at 201C), washed 3 in PBS conjugated second antibody. and 3 in PBSþ and AlexaFluor-488-conjugated donkey anti-rat Fibres were examined using a Zeiss LSM 510 confocal micro- antibody (1:1000 dilution; 1 h; 41C). The slides were washed 3 in scope using a  100 (1.4 NA) lens, labelled tracks measured using PBS and 3 in PBSþ , and IdU detected using BrdU anti-sheep the LSM software (white bars on individual images show examples antibody (1:1000 dilution; 15 h at 41C). Slides were then washed of measurements recorded) and converted to kilobase pairs using a 3 in PBS, 3 in PBSþ and incubated with Cy3-conjugated conversion factor of 1mm¼ 2.59 kb (Jackson and Pombo, 1998). donkey anti-sheep antibody (1:1000 dilution; 1 h at 41C). Finally, Measurements were recorded in randomly selected fields (selected the slides were washed 3 in PBSþ,3 in PBS, incubated with at low power) from dispersed, untangled areas of the DNA spread. 5mg/ml Hoechst 33258 (Sigma) for 10 min, rinsed 3 in PBS and As the analysis of single, unbroken fibres is a key feature of this mounted with 50:50 PBS–glycerol. study, routine quality control for spreading of different cell types For nuclei labelled with Cy3-dUTP and/or AlexaFluor-488- under different experimental conditions was performed using direct conjugated dUTP, slides were washed 3 in PBS, incubated with DNA labelling with YOYO (Merrick et al, 2004) or BrdU 5mg/ml Hoechst 33258 (Sigma) for 10 min, and then rinsed 3  in immunolabelling of fibres from cells labelled with BrdU for B24 h. PBS and mounted with PBS–glycerol. Slides were examined using a Zeiss LSM 510META confocal Replication foci in situ microscope using a  100(1.4 NA) lens. 3D images were generated Modified nucleoside triphosphates were introduced into cells using TM using Z stacks and processed in Imaris software. FuGene (Roche). Briefly, DT40 cells were pelleted (200 g, 4 min), washed 3  in cold PBS and 110 cells in 10ml PBS transfected by incubating with transfection mix (12ml PBS, 3ml of FuGene and 1ml Supplementary data (1 nmol) of the required dUTP analogue) for 10 min at 01C. After Supplementary data are available at The EMBO Journal Online transfection, the cells were washed in fresh medium (0.5 ml of (http://www.embojournal.org). medium, 200 g, 4 min) and incubated in medium (0.5 ml) for 30 min at 371C. For double labelling, the same protocol was used for the second dUTP analogue. For experiments using BrdU, cultures were Acknowledgements pulse labelled with 25mM BrdU either before or after transfection. HeLa cells were grown on microscope coverslips and transfected This work was funded by the BBSRC (linked project grant BBS/B/ with biotin-11-dUTP. Coverslips were rinsed in PBS and placed over 06091 to DAJ and KWC). We thank Chi Tang and other colleagues a 7.5ml droplet of transfection mix on parafilm and incubated for for technical advice and support. References Berezney R, Dubey DD, Huberman JA (2000) Heterogeneity of Jackson DA, Pombo A (1998) Replicon clusters are stable units of eukaryotic replicons, replicon clusters, and replication foci. chromosome structure: evidence that nuclear organization con- Chromosoma 108: 471–484 tributes to the efficient activation and propagation of S phase in Blow JJ, Gillespie PJ, Francis D, Jackson DA (2001) Replication human cells. J Cell Biol 140: 1285–1295 origins in Xenopus egg extracts are 5–15 kb apart and are acti- Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. vated in clusters that fire at different times. J Cell Biol 152: 15–26 Nature 432: 316–323 Fisher D, Mechali M (2004) Sleeping policemen for DNA replica- Kilbey A, Black EJ, Unlu M, Gillespie DAF (1996) The v-Jun tion? Nat Cell Biol 6: 576–577 oncoprotein replaces p39 c-Jun as the predominant AP-1 consti- Gilbert DM (2002) Replication timing and transcriptional control: tuent in ASV17-transformed fibroblasts: implications for SAPK/ beyond cause and effect. Curr Opin Cell Biol 14: 377–383 JNK-mediated signal transduction. Oncogene 12: 2409–2418 Jackson DA (1995) Nuclear organization—uniting replication foci, Lam H, Liu Q, Elledge SJ, Rosen JM (2004) Chk1 is haploinsufficient chromatin domains and chromosome structure. Bioessays 17: for multiple functions critical to tumor suppression. Cancer Cell 6: 587–591 45–59 2730 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Shechter D, Gautier J (2005) ATM and ATR check in on origins—a Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge dynamic model for origin selection and activation. Cell Cycle 4: SJ (2000) Chk1 is an essential kinase that is regulated by Atr and 235–238 required for the G(2)/M DNA damage checkpoint. Genes Dev 14: Sorensen CS, Syljuasen RG, Falck J, Schroeder T, Ronnstrand L, 1448–1459 Khanna KK, Bartek J, Lukas J (2003) Chk1 regulates the S phase Machida YJ, Hamlin JL, Dutta A (2005) Right place, right time, and checkpoint by coupling the physiological turnover and ionizing only once: replication initiation in metazoans. Cell 123: 13–24 radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell Miao H, Seiler JA, Burhans WC (2003) Regulation of cellular and 3: 247–258 SV40 virus origins of replication by Chk1-dependent intrinsic and Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, UVC radiation-induced checkpoints. J Biol Chem 278: 4295–4304 Johansson F, Helleday T, Sehested M, Lukas J, Bartek J (2005) Marheineke K, Hyrien O (2004) Control of replication origin Inhibition of human Chk1 causes increased initiation of DNA density and firing time in Xenopus egg extracts—role of a replication, phosphorylation of ATR targets, and DNA breakage. caffeine-sensitive, ATR-dependent checkpoint. J Biol Chem 279: Mol Cell Biol 25: 3553–3562 28071–28081 Takai H, Tominaga K, Motoyama N, Minamishima YA, Nagahama Merrick CJ, Jackson D, Diffley JFX (2004) Visualization of altered H, Tsukiyama T, Ikeda K, Nakayama K, Nakanishi M, Nakayama replication dynamics after DNA damage in human cells. J Biol K (2000) Aberrant cell cycle checkpoint function and early Chem 279: 20067–20075 embryonic death in Chk1(/) mice. Genes Dev 14: 1439–1447 Norio P, Kosiyatrakul S, Yang QX, Guan ZQ, Brown NM, Thomas S, Woodward AM, Go¨hler T, Luciani MG, Oehlmann M, Ge X, Gartner Riblet R, Schildkraut CL (2005) Progressive activation of DNA A, Jackson DA, Blow JJ (2006) Excess Mcm2–7 license dormant replication initiation in large domains of the immunoglobulin origins of replication that can be used under conditions of heavy chain locus during B cell development. Mol Cell 20: replicative stress. J Cell Biol 173: 673–683 575–587 Zachos G, Rainey MD, Gillespie DAF (2003) Chk1-deficient tumour Petermann E, Maya-Mendoza A, Zachos G, Gillespie DAF, Jackson cells are viable but exhibit multiple checkpoint and survival DA, Caldecott KW (2006) Chk1 requirement for high global rates defects. EMBO J 22: 713–723 of replication fork progression during normal vertebrate S phase. Zachos G, Rainey MD, Gillespie DAF (2005) Chk1-dependent S–M Mol Cell Biol 26: 3319–3326 checkpoint delay in vertebrate cells is linked to maintenance of Sadoni N, Cardoso MC, Stelzer EHK, Leonhardt H, Zink D (2004) viable replication structures. Mol Cell Biol 25: 563–574 Stable chromosomal units determine the spatial and temporal Zhao H, Watkins JL, Piwnica-Worms H (2002) Disruption of the organization of DNA replication. J Cell Sci 117: 5353–5365 checkpoint kinase 1/cell division cycle 25A pathway abrogates Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Molecular mechanisms of mammalian DNA repair and the DNA Sci USA 99: 14795–14800 damage checkpoints. Annu Rev Biochem 73: 39–85 Zhang JM, Xu F, Hashimshony T, Keshet N, Cedar H (2002) Shechter D, Costanzo V, Gautier J (2004) ATR and ATM regulate the Establishment of transcriptional competence in early and late S timing of DNA replication origin firing. Nat Cell Biol 6: 648–655 phase. Nature 420: 198–202 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2731 | | http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The EMBO Journal Springer Journals

Chk1 regulates the density of active replication origins during the vertebrate S phase

Download PDF
13 pages

Loading next page...
 
/lp/springer-journals/chk1-regulates-the-density-of-active-replication-origins-during-the-tLKWCMngUr

References (30)

Publisher
Springer Journals
Copyright
Copyright © European Molecular Biology Organization 2007
ISSN
0261-4189
eISSN
1460-2075
DOI
10.1038/sj.emboj.7601714
Publisher site
See Article on Publisher Site

Abstract

The EMBO Journal (2007) 26, 2719–2731 & 2007 European Molecular Biology Organization All Rights Reserved 0261-4189/07 | | THE THE www.embojournal.org EMB EMB EMBO O O JO JOU URN R NAL AL Chk1 regulates the density of active replication origins during the vertebrate S phase 1 2 these sub-chromosomal units is thought to play a direct role Apolinar Maya-Mendoza , Eva Petermann , 3 2 in defining the S-phase programme (Sadoni et al, 2004). David AF Gillespie , Keith W Caldecott 1, In human cells, DNA is duplicated within an S phase of and Dean A Jackson * about 10 h and during this time, roughly 50 000 replication Faculty of Life Sciences, University of Manchester, MIB, Manchester, origins are used to activate synthesis of replicons that are UK, Genome damage and Stability Centre, University of Sussex, Falmer, typically B100 kb in length (Jackson and Pombo, 1998; Brighton, UK and Beatson Institute for Cancer Research, Cancer Research UK, Glasgow, UK Berezney et al, 2000). Potential origins are activated through- out S phase so that B10% of replicons are engaged in DNA The checkpoint kinase 1 (Chk1) preserves genome integ- synthesis at any time. Single-molecule analysis of DNA fibres rity when replication is performed on damaged templates. shows that specific origins or groups of origins function at Recently, Chk1 has also been implicated in regulating specific times of S phase and can be developmentally regu- different aspects of unperturbed S phase. Using mamma- lated (Jackson and Pombo, 1998; Norio et al, 2005). In lian and avian cells with compromised Chk1 activity, we addition, the temporal activation of specific regions of the show that an increase in active replicons compensates for genome correlates with the appearance of distinct patterns of inefficient DNA polymerisation. In the absence of damage, replication sites as cells progress from early to middle and loss of Chk1 activity correlates with the frequent stalling then late S phase (reviewed in Jackson, 1995). and, possibly, collapse of active forks and activation of In somatic vertebrate cells, almost nothing is known about adjacent, previously suppressed, origins. In human cells, the mechanisms that regulate the temporal activation and super-activation of replication origins is restricted to pre- suppression of potential replication origins (Gilbert, 2002; existing replication factories. In avian cells, in contrast, Machida et al, 2005). However, recent studies have suggested Chk1 deletion also correlates with the super-activation of that the replication checkpoint pathways contribute to replication factories and loss of temporal continuity in the the regulation of origin activation during normal S phase replication programme. The same phenotype is induced in (reviewed in Fisher and Mechali, 2004; Kastan and Bartek, wild-type avian cells when Chk1 or ATM/ATR is inhibited. 2004). Although checkpoint pathways are activated in re- These observations show that Chk1 regulates replication sponse to DNA damage (reviewed in Sancar et al, 2004), a origin activation and contributes to S-phase progression in function in unperturbed S phase is implied by the fact that somatic vertebrate cells. checkpoint kinase 1 (Chk1) is essential for normal develop- The EMBO Journal (2007) 26, 2719–2731. doi:10.1038/ ment (Liu et al, 2000; Takai et al, 2000). In the absence of sj.emboj.7601714; Published online 10 May 2007 DNA damage, Chk1 has been shown to regulate the physio- Subject Categories: cell cycle; genome stability & dynamics logical turnover of Cdc25A, which in turn regulates the Keywords: checkpoint proteins; DNA foci; DNA replication; activation of replication origins through the action of CDK2, replication origins; S-phase programme associated A and E type cyclins and Cdc45 (Zhao et al, 2002; Sorensen et al, 2003). This pathway, in principle, provides a mechanism for regulating origin activation throughout S phase (Fisher and Mechali, 2004; Shechter and Gautier, 2005), although how a spatially ordered programme of origin activation might be established remains unclear. Introduction In this report, we have used cells with compromised ATM/ ATR and Chk1 function to explore how the checkpoint path- During cell proliferation, eukaryotic genomes are replicated ways contribute to the local and long-range activation of DNA with high fidelity in order to ensure their genetic integrity. synthesis. We have recently shown that Chk1 is required to Because of their size, the duplication of eukaryotic genomes maintain normal rates of replication fork progression in demands that DNA synthesis is activated at numerous repli- human and avian cells in the absence of DNA damage cation ‘origins’ (Machida et al, 2005). In mammalian cells, (Petermann et al, 2006). In the present study, we have replication origins are activated in small groups (Jackson and analysed how ATM/ATR and Chk1 influence the local activa- Pombo, 1998; Berezney et al, 2000) that are replicated tion of replication origins within replicon clusters and the together within individual replication factories. These repli- spatial organisation of replication foci throughout S phase. con clusters can be visualised as DNA foci and the sequential When Chk1 function is compromised, a 2- to 3-fold increase activation of potential replication origins within groups of in origin density is seen, showing that Chk1 normally func- tions to regulate the density of active origins by suppressing *Corresponding author. Faculty of Life Sciences, University of many of the potential origins that could be activated within Manchester, MIB, 131 Princess Street, Manchester M1 7DN, UK. Tel.: þ 1 0161 306 4255; Fax: þ 1 0161 306 8918; each replicon cluster. In human cells, this super-activation of E-mail: [email protected] replication origins in cells with reduced Chk1 activity is restricted to replicons that are already associated with active Received: 24 October 2006; accepted: 17 April 2007; published replication sites, suggesting that the checkpoint proteins must online: 10 May 2007 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2719 | | Chk1 regulates origin density A Maya-Mendoza et al function in the context of spatial constraints that are defined consistent with Chk1 contributing to the regulation of repli- by the programmed assembly of replication factories. cation origin activation (Miao et al, 2003; Syljuasen et al, 2005). However, formal proof for the super-activation of replication origins was not presented in these studies. Results Using spread DNA fibres (Jackson and Pombo, 1998), we first mapped the structure of active replicons in cells that are Replicon structure Cells with perturbed Chk1 function display a small global deficient for ATR and Chk1 function (Figures 1 and 2). Active increase in DNA synthesis and chromatin-associated Cdc45, replicons were labelled with biotin-dUTP following introduc- A B Figure 1 Chk1 regulates the density of active replication origins in HeLa cells. Replication structures were visualised on spread DNA fibres (A–F) using biotin incorporation (shown in red) to assign replication fork polarity. After spreading and immunolabelling, replicon structure was defined by the distance between (panel A) adjacent origins in replicon clusters and (panel B) sister replication forks. Changes in replicon structure induced by treatment with caffeine (panels C–E) and UCN-01 (panels C, F) were determined. Frequency histograms (panels A–C) show the distribution of separation in kilobase pairs, assigned using spreads like the typical examples shown (panels A, B, D–F). Parameters defining average replicon structure under different conditions are shown (G). To monitor the affect of checkpoint protein inhibitors on replication foci (H), replication sites were pulse labelled for 20 min with BrdU (green) and then for 25 min with IdU (red). Unsynchronised cultures were treated with caffeine or UCN-01 before and throughout first labelling and S-phase cells assigned to early, mid or late S phase. Images are merges of confocal sections, with areas of colocalisation shown in yellow. Scale bars (5mm) are shown on individual images. 2720 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al AB Figure 2 Chk1 regulates replication origin density in diploid human fibroblasts. Replication structures of MRC5 human fibroblasts were visualised using the procedures described in Figure 1. Replicon size (A–D) was measured using the separation of sister forks (panel A) after labelling with biotin-dUTP (panels B–D) and structures in untreated cells compared with those in cells treated with caffeine or UCN-01. BrdU labelling was also used to determine the rate of fork elongation (E–G) under the same conditions. Parameters defining average replicon structure under different conditions are shown (H). The organisation of active replication foci in cells treated with caffeine and UCN-01 was compared with untreated cells (I), as described in Figure 1. Scale bars, 5mm. tion of a very low concentration of precursor into cells. As the labelled tracks allows unambiguous assignment of the daugh- precursor is consumed during the labelling period, immuno- ter forks that emanate from a common, central replication detection of biotin reveals comet-like labelled tracks, where origin (Figure 1A and B). the head of the comet represents the position of replication at As the majority of replicons in vertebrate cells are co- the beginning of the labelling pulse and the tail the point replicated in small replicon clusters, the origin spacing within when the label was consumed. Hence, the structure of these clusters provides a direct measure of the replicon length &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2721 | | Chk1 regulates origin density A Maya-Mendoza et al (Figure 1A). In unsynchronised HeLa cells, adjacent origins synthesis was profoundly altered in the presence of caffeine were, on average, 159 kb apart (Figure 1G), similar to the at the level of individual forks (Figure 1A–G), no defects were value of 145 kb during early S phase (Jackson and Pombo, seen at the level of changes in global sites of DNA synthesis. 1998). Although inter-origin distance measurements are robust, Hence, in the presence of inhibitor, colocalisation of the two this analysis neglects isolated replicons and the outgrowing labels and the maintenance of replication patterns with the forks of replicon clusters, which can be much longer than normal number and structure of replication foci shows that those that are located between the active origins (Berezney inhibitor-induced super-activation of latent origins was re- et al, 2000). To incorporate these forks, we also measured the stricted to replicons that were engaged with active replication separation of the daughter forks of individual replicons factories (Figure 1H). Following the most effective caffeine (Figure 1B). As the cells are unsynchronised, labelled repli- treatment, almost all active centres contained the first and cons are visualised at steady state so that the average second labels (Po0.47 (n ¼ 26) using Student’s t-test to separation of daughter forks at the time of labelling will compare control and caffeine-treated cells). approximate to half the replicon length. In unsynchronised The super-activation of latent origins seen in HeLa cells cells, the expected wide range of daughter fork separations was confirmed in MRC5 cells, which are normal human was seen, with an average of 72 kb (Figure 1B and G), diploid fibroblasts from lung (Figure 2). These diploid fibro- roughly half the average inter-origin spacing. blasts have a more uniform origin spacing and average The distribution of daughter forks was also assessed in separation that is about half that in transformed HeLa cells. unsynchronised cells that had been treated with the kinase Even so, DNA spreads from control and caffeine- or UCN-01- inhibitors caffeine-general inhibitor ATR/ATM kinase activ- treated MRC5 cells (Figure 2A–H) showed a clear 2- to 3-fold ity—and UCN-01—a specific inhibitor of Chk1 autophosphory- increase in origin activation (Figure 2A–D) and a correspond- lation (Figure 1C–G). When caffeine was added either 30 min ing fall in the rate of fork migration (Figure 1E–H) upon before the time of replication labelling (designated 30 caf- inhibition of the ATR/ATM pathways. As for HeLa cells feine) or 30 min before and throughout labelling (Figure 1), the observed super-activation of latent origins in 0 0 (designated 30 –30 caffeine; Figure 1C) a clear approximately MRC5 fibroblasts was restricted to pre-established replication 2- to 3-fold decrease in fork separation was seen (Figure 1G). factories (Figure 2I). Similar results were observed when the Chk1 inhibitor UCN-01 was added 60 min before and throughout labelling, with Caffeine-induced activation of latent replication origins replication structures B2-fold closer than in untreated cells We next used a double labelling strategy to define the (Figure 1C and G); slight differences in the caffeine- and UCN-01- relationship between origins that were induced in response induced responses may imply that ATM/ATR also inhibit fork to replication stress and the previously active forks movement through other mechanisms that are independent (Figure 3). Cells were preincubated in caffeine and pulse of Chk1. In both cases, these increases in origin density are labelled for 20 min with bromo-deoxyuridine (BrdU) in pre- conservative estimates because the analysis monitors paired sence of caffeine, washed and then pulse labelled with biotin- sister forks but does not score conjoined forks from origins dUTP (Figure 3A). This labelling protocol defines five classes that initiated synthesis during the labelling pulse. of replication structure (Figure 3B). For both HeLa and MRC5 A notable feature of this experiment is that the increased (Figure 3C), B67% of forks were labelled consecutively with origin density results in a clear decrease in the proportion of the first and second labels (class 1), as expected for forks that widely spaced forks. While cells must contain the natural were elongating throughout the labelling period. In both cell distribution of forks at the beginning of this experiment, the types, 16% of labelled structures contained two forks grow- relative decrease in remote daughter fork results from two ing from a common origin (class 2) and a similar number of factors: (1) novel initiation events dominate the analysis growing forks fused and so terminated during labelling (class 3). because of the high frequency of caffeine-induced origin In controls, very few tracks were labelled uniquely with the activation. These recently activated origins with close sister first or second label (class 4). forks (Figure 1E and F) represent the major population seen In the presence of caffeine, the rate of fork elongation was in the randomly selected fields analysed and (2) widely reduced by at least two-fold throughout the labelling period separated forks are inevitably from long replicons, with (compare control and caffeine class 1 forks in Figure 3B). In replication complexes engaged throughout the labelling pro- addition, quite different proportions of replicating structures tocol. Reduced efficacy of replication at some forks compro- were seen in caffeine treated samples (Figure 3C). Strikingly, mises the structure and assignment of these forks as bona the proportion of consecutively labelled forks (class 1) fell by fide sister pairs. Even so, if spreads are scanned using biased 43-fold as a result of two factors. First, caffeine treatment selection criteria, the active forks in long replicons can be resulted in a 42-fold increase in the number of new initiation found. events during both the first (class 2) and second (class 4) labelling periods. Second, a dramatic increase in the fre- Global analysis of replication in caffeine-treated cells quency of closely spaced active origins (class 5) was also We next characterised how defects in the ATM/ATR pathways seen. Despite a clear fall in the proportion of elongation forks, influence the global structure of replication sites, in order to o5% of forks (class 4—green only) stalled during the first evaluate if the initiation events induced by caffeine and pulse and remained inactive during the second. These ob- UCN-01 were distributed throughout the nucleus. Changes servations show that in the presence of caffeine, active forks in the structure of the replication programme were assessed frequently stall and sometimes collapse, and that these defects using simple labelling and indirect immunofluorescence in replication are compensated for by the activation of novel, (Supplementary Figure S1), and double labelling caffeine-dependent replication origins. A diagnostic feature of (Figure 1H). This analysis demonstrates that whereas DNA novel origin activation is the dramatic increase in interspersed 2722 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al Figure 3 Activation of latent origins in human cells treated with caffeine. HeLa and MRC5 cells were treated with caffeine and pulse labelled with BrdU and biotin-11-dUTP, as shown (A). DNA fibres were prepared, treated with acid and immunolabelled to visualise BrdU (green) and biotin-dU (red). Five classes of replication structure were defined (B; classes 1–5) and the relative frequency of occurrence of the different classes was scored (frequency histograms (C)). Scale bars, 5mm. labelling (class 5). Figure 3B (class 5 caffeine) shows a typical Detailed analysis of replicon structure in DT40 cells extreme case with a 75 kb region containing four active showed that an increased origin density compensates for origins. Clearly, this origin density is very much higher than the reduced fork rate (Figure 4). Replicons of wild-type (wt) is normally seen in control cells (Figures 1 and 2). DT40 cells were smaller than those of HeLa cells, with In the presence of caffeine, this increased density of active an average daughter fork separation of 36.7 kb and origin forks, together with a high frequency of clustered caffeine- spacing of 67.8 kb (Figure 4). Replicons in Chk1-deficient dependent initiation events in the vicinity of previously active DT40 cells were even smaller than those in wt control cells forks, confirms that the vast majority of novel initiation (Po2.7E-19), with an average daughter fork separation of events occur in already active replicon clusters. Under these 24.2 kb and average origin spacing of 47.3 kb. This represents conditions, the efficacy of replication is severely compro- a 1.5- to 2-fold increase in the number of active origins in mised, with all forks showing reduced rates of elongation, Chk1-deficient DT40 cells, which is sufficient to maintain the consistent with frequent stalling during synthesis. Despite normal S-phase duration when individual forks polymerise this reduced rate, a majority of forks continue synthesis and nucleotides at B50% of the rate in wt cells (Petermann et al, only a minority collapse (Figure 3). Importantly, this experi- 2006). ment shows that active replicons contain latent origins ahead As for HeLa and MRC5 cells, clear reductions in daughter of the growing fork, which are suppressed under normal fork separation were seen in DT40 cells treated with UCN-01 conditions but activated in response to replicative stress. (Po2.52E-12) or caffeine (Po1.19E-21) (Figure 4B and D); similar reductions in fork to fork distance were seen follow- Organisation of DNA replication in Chk1 cells ing treatment with caffeine and UCN-01 (Po0.43). A very Because inhibitors such as caffeine might alter or stress the similar replicon structure was also seen in Chk1 DT40 normal pathways that regulate the activation of replication cells (Po0.548 Chk1 versus wt DT40 incubated with origins, we next wanted to analyse if DNA synthesis was caffeine; Figure 4C and D). This shows that DT40 cells have perturbed in Chk1-deficient cells. For this purpose, we chose a very similar density of active replication origins when either to use Chk1-deficient DT40 (Zachos et al, 2003), as Chk-1 ATR/ATM or Chk1 function is compromised and implies that depletion in mammalian model systems is known to be cell Chk1 plays a major role in regulating active origin density in lethal (Liu et al, 2000). Previously, we showed that Chk1- these cells. deficient DT40 cells have a reduced rate of replication at individual forks (Petermann et al, 2006), which appears The replication programme in DT40 cells contrary to the observed increase in global DNA synthesis Defects in origin activation in Chk1-deficient DT40 cells when UCN-01 was used to disrupt Chk1 function in mamma- prompted us to evaluate how the absence of Chk1 influenced lian cells (Syljuasen et al, 2005). the S-phase programme in these cells. As for HeLa cells &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2723 | | Chk1 regulates origin density A Maya-Mendoza et al A BC DT40 chk1–/– D DT40 chk1–/– cells E G DT40 Chk1–/– DT40 Chk2–/– Figure 4 Inhibition of ATR or Chk1 mimics the replication phenotype of Chk1-deleted DT40. Active replicons in Chk1 and isogenically matched wt DT40 cells were analysed after pulse labelling with biotin-11-dUTP. Replication structures were visualised on spread DNA fibres as in Figure 1. Changes in replicon structure in cells with compromised Chk1 activity are shown (frequency histograms (A–C)). In wt DT40, caffeine and UCN-01 (panel B) produced a significant decrease in replicon size (Po1.197E-21 and 2.52E-12, respectively, versus controls). No difference was seen when Chk1 DT40 cultures (panel C) were treated with caffeine (P¼ 0.541). Parameters defining average replicon structure under different conditions are shown (D). The S-phase programme of wt DT40 was also compared with Chk1- and Chk2-deficient cells. Cells were pulse labelled for 20 min with BrdU, immunolabelled (E) and the proportion (F) of early-, mid- and late-S-phase patterns determined. Patterns that did not conform to the recognised structural and spatial criteria were designated as mixed. Caffeine-induced changes in the organisation of replication foci (G) were assessed by double labelling, as described in Figure 1H. High-power details are from the boxed areas shown. Scale bars (5 and 0.5mm in Detail) are shown on individual images. (Figure 1), the S-phase programme of wt DT40 cells (Zachos and Supplementary Figure S2). Chk2-deficient DT40 cells, in et al, 2003) can be described by the size, complexity and contrast, displayed the normal distribution of replication nuclear distribution of their replication foci (Figure 4E–G and patterns, with no significant changes in the structure or Supplementary Figure S2). Using wt DT40, patterns that are number of replication foci (Figure 4E and Supplementary characteristic for this cell type were seen using different Figure S2). These observations imply that the natural link labelling protocols, which allow multiple labelling without between DNA synthesis and the spatial organisation of active reagent cross-talk in fixed or living cells (Figure 4E and replication foci is compromised in Chk1-deficient DT40 cells. Supplementary Figure S2). Chk1-deficient cells had the same range of replication structures as parental DT40 con- Defects in the DT40 replication programme trols. However, it was immediately obvious that the complex- Quantitative analysis of early-, mid- and late-S-phase patterns ity of foci in these cells was greater than in controls. In early showed that Chk1-deficient cells had a higher proportion of S phase, high-resolution 3D analysis of replication foci showed mixed S-phase structures than wt controls (Figure 4F). To DT40 cells to have 3907176 (n ¼ 10) foci, whereas Chk1- extend this observation, we used double labelling protocols deficient cells, with 7557425 (n ¼ 7) foci, had roughly two- to describe the spatial architecture of the S-phase programme fold more active sites. The replication patterns in Chk1 (Figure 4G). The vertebrate S phase is structured so that the and wt cells were shown to be statistically different using the sequential activation of replicon clusters occurs at spatially Mann–Whitney test (Po0.02; U-value 59). Notably, the foci adjacent sites as S phase proceeds (Sadoni et al, 2004). This in Chk1-deficient cells were smaller and more homogeneous spatial relationship is maintained in wt DT40 (Figure 4G), in structure, independently of labelling intensity (Figure 4E where 8177% of foci showed colocalisation when the two 2724 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al pulse labels were applied consecutively. Notably, when dou- of B250 kb, with two adjacent active replicons, were ana- ble labelling was performed in Chk1-deficient cells, colocali- lysed. Typical fibres from DT40 cells displayed four active sation was only seen at 59716% of foci (Figure 4G, Detail forks that were each labelled throughout both the first and and Supplementary Figure S2D), which was very similar to second pulses (Figure 5C). The two origins in this cluster are the colocalisation in both wt and Chk1 DT40 treated with 32.84 mm (85 kb) apart and all four of the active forks show caffeine (Supplementary Figure S2D). Hence, Chk1-deficient similar rates of elongation (Figure 5C). Figure 5D shows a cells show a 42-fold increase in the number of active foci similarly labelled DNA fibre from Chk1-deficient cells, in that are spatially uncoupled from foci that were labelled which an additional origin (þ ) was activated between the immediately upstream in the replication programme. From two already active origins, during the second pulse label. these experiments, we conclude that Chk1 contributes to the Such initiation within B10 kb of two converging forks is mechanism that normally regulates S-phase progression in extremely uncommon in wt cells, but not unusual, if Chk1 DT40 cells. function is perturbed (e.g. Figures 1F and 5A) and is con- Following treatment with caffeine, the structure of replica- sistent with a role of Chk1 in regulating the activation of tion foci in wt DT40 cells mimicked those seen in Chk1- potential replication origins during the normal S phase. deficient cells (Figure 4G). After 50 min in the presence of caffeine, a B2-fold increase in the complexity of early- S-phase foci was seen, although no increase was seen when Aberrant initiation events in Chk1-deficient cells are Chk1-deficient cells were treated with caffeine (Figure 4G and linked to defects in active replication forks Supplementary Figure S3). Similar observations were re- We next explored if aberrant initiation events in cells with corded in cells treated with UCN-01 (Supplementary Figure compromised Chk1 activity were related to intrinsic defects S3). As for Chk1 DT40, double labelling of replication foci in DNA replication (Syljuasen et al, 2005; Petermann et al, in DT40 cells treated with Chk1 inhibitors showed a B2-fold 2006). Normally, BrdU in DNA is only detected by immuno- increase in foci that were not colocalised during consecutive labelling if DNA is first denatured (typically by HCl treat- pulse labels (Figure 4G, Detail and Supplementary Figure S2). ment). However, BrdU incorporated into the DNA of Chk1- This is consistent with disorganisation of the replication deficient cells is readily detectable without denaturation and programme in the presence of Chk1 inhibitors and shows TUNEL analysis confirms that this is due to persistent DNA that caffeine and UCN-01 phenocopy Chk1 deletion in DT40 nicks (Syljuasen et al, 2005; Petermann et al, 2006). cells. Cells were pulse labelled consecutively with BrdU and biotin-dUTP and labelled tracks of spread DNA fibres visua- Unprogrammed initiation of DNA synthesis lised either without or with prior acid treatment (Figure 6 and in Chk1 DT40 Supplementary Figure S5). When immunolabelling was per- To look more precisely at the specific roles of Chk1, we also formed after acid treatment, 490% of forks had adjacent analysed if the activation of DNA synthesis was perturbed in tracks labelled with BrdU and biotin-dU, in both wt and Chk1-deficient DT40 cells under normal—unstressed— Chk1-deficient cells (Figure 6; (þ )HCl). However, when growth conditions (Figure 5). Cells were labelled for 24 h acid treatment was omitted, incorporated BrdU was almost with BrdU before pulse labelling with biotin-dUTP. After completely undetected in spreads from wt cells, with o2% of spreading, both analogues were visualised by indirect immuno- biotin tracks showing very weak (background) BrdU immu- labelling and the distribution of biotin-labelled replication nostaining (Figure 6A, ()HCl). In contrast, 16% of biotin forks was analysed in single, unbroken DNA fibres of tracks in spreads from Chk1-deficient cells had adjacent B250 kb. In a typical experiment (Figure 5A and B), labelled regions of BrdU immunostaining (Figure 6B, ()HCl and regions of isolated (single), unbroken DNA strands were Supplementary Figure S5). These labelled regions were typi- selected at random (using BrdU labelling) and aberrant cally 2.5–10 kb long and the fragmented labelling was clearly replication structures within 25 kb of the paired forks of different in structure to the typical B25 kb tracks seen after replicon clusters with two active replicons were recorded HCl treatment. Notably, the acid-independent BrdU labelling (Figure 5A). In wt DT40, 93.5% of labelled replicons con- in Chk1-deficient cells was frequently adjacent to a newly formed to the expected structure, with pairs of divergent activated origin. A typical example is shown (Figure 6B, sister forks. Only 6.5% had any associated unexpected label- ()HCl and bottom diagram in 6C), where fragmented ling (n ¼ 46; average length of labelled region 93.9 kb). In BrdU labelled overB10 kb lies adjacent to a pair of divergent, contrast, 35% of randomly selected replication structures biotin-labelled forks. Clearly, these biotin-labelled forks from Chk1-deficient DT40 (Figure 5A and B) had associated resulted from the activation of a novel replication origin aberrant replication (n ¼ 40; average length of labelled region towards the end of the BrdU labelling period, at a position 56.5 kb). that is B12 kb from the boundary of the BrdU labelling. As This B5-fold increase of aberrant replication structures in the DNA adjacent to the BrdU boundary is labelled with Chk1-deficient cells is consistent with the activation of novel biotin, the boundary region must represent a termination site, origins, which normally would only occur under conditions where the biotin-labelled and stalled or aberrant BrdU- of replicative stress (Woodward et al, 2006). To emphasise labelled forks met (Figure 5C). this point, we developed a triple labelling protocol, which This experiment makes two key points: (a) that replication provides a more precise description of active replicon struc- forks in Chk1-deficient cells are intrinsically unstable and ture (Figure 5). Cells were labelled for 24 h with BrdU and prone to accumulate ssDNA breaks over many kilobase pulse labelled sequentially with digoxigenin-dUTP and biotin- pairs and (b) that at some forks this damage correlates dUTP, and spread DNA fibres were immunolabelled (Figure 5C with the activation of latent origins that typically are located and D and Supplementary Figure S4). Undamaged DNA fibres 10–20 kb away. &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2725 | | Chk1 regulates origin density A Maya-Mendoza et al Structure of replicons and DNA foci in chick embryonic cells were treated with inhibitors of the checkpoint pathways fibroblasts as describe before. As for human and DT40 cells, CEFs grown As checkpoint pathways may be perturbed in cells, such as in caffeine or UCN-01 showed a B2-fold increase in replica- DT40, that are adapted to long-term culture, we next con- tion fork density, consistent with increased origin activation firmed the organisation of replicons and DNA foci in primary when Chk1 function was inhibited (Figure 7A and C). This avian cells. Early-passage chick embryonic fibroblasts (CEF) increased origin density occurred in cells with B50% of the DT40 wt n=46 DT40 Chk–/– n=46 2nd 1st pulse 2nd 2nd 1st pulse 2nd 2726 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al natural rate of fork elongation (Figure 7B and C). As with Discussion DT40 cells (Figure 4), in the presence of caffeine and UCN-01, CEFs showed a significant (B2-fold) increase in the number Under conditions of replicative stress, eukaryotic cells ex- of their active replication foci (Figure 7D and E and press sophisticated checkpoint pathways that serve to ensure Supplementary Figure S2), which were spatially disorganised the preservation of genomic integrity. If DNA is damaged relative to untreated cells (Figure 7F). during S phase, activation of the intra-S-phase checkpoint A B (–) HCI (–) HCI Merge Merge (–) HCI (–) HCI Merge Merge (–) HCI Biotin (–) HCI BrdU (+) HCI Merge (+) HCI Merge (+) HCI (+) HCI (–) HCI (+) HCI (–) HCI Figure 6 Novel origin activation is spatially coupled to aberrant replication structures in Chk1 DT40 cells. wt and Chk1-deficient DT40 cells were pulse labelled consecutively with BrdU (20 min) and biotin-dUTP (30 min). Spread DNA fibres were immunolabelled to visualise sites of incorporation of BrdU (green) and biotin-dU (red). Labelling was performed either with or without prior treatment with HCl, as shown (A, B). Merged images show overlays of green and red channels (panels A, B). In panel B, the merged image and individual channels are shown for a high-power view of a single fibre. A diagram describing the analysis is shown (C), using the nomenclature described in Figure 5. X indicates a boundary between regions labelled in the first pulse and a nearby replicon that was labelled predominantly during the second, as seen in panel B (high-power example). Scale bars (5 or 10mm) are shown on individual images. Figure 5 Unprogrammed activation of DNA synthesis in Chk1-deficient DT40 cells. Chk1 and isogenically matched wt DT40 cells were labelled with BrdU for 24 h and pulse labelled with biotin-dUTP (30 min; (A, B)) or Dig-dUTP, followed by biotin-11-dUTP (both 30 min; (C, D)). In panel (A) are shown sites of biotin-dU incorporation (red) in single, isolated DNA fibres from wt and Chk1 DT40 cells. DNA fibres were labelled with BrdU to ensure that only single fibres were used for the analysis; for simplicity the BrdU labelling is not shown (some examples are provided in Supplementary Figure S4 and S5). Replicon structure was defined by the organisation of the growing forks (diagram (panel A); filled triangles represent the direction of fork growth from central origins, depicted by open arrows; þ represents a novel or aberrant replication structure) using fibres with two active replicons (four labelled forks). The relative frequency of normal (bidirectional) and aberrant replication structures (within 25 kb of normal forks) was scored (panel B). Unprogrammed initiation in Chk1 cells was confirmed using a combination of Dig-dU and biotin-dU to define replicon structure (panels C, D). DNA fibres were immunolabelled to visualise isolated fibres with two active origins (B250 kb DNA). Such replicons contain two pairs of growing forks that were labelled first with Dig and then with biotin (panels C, D). In Chk1 DT40 (panel D), but not in wt controls (panel C), unprogrammed initiation contributes to synthesis of the unreplicated DNA between the active growing forks—in this example, a new initiation event ( þ ) occurred in the centre of the fibre during the second pulse. Diagrams of the structure of the labelled replicons are shown (panels C, D), using nomenclature described in panel A. Scale bars (5mm (panel A) and 10mm (panels C, D)). &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2727 | | Chk1 regulates origin density A Maya-Mendoza et al AB 30′ caffeine 30′–30′ caffeine 30′–20′ caffeine 5 mM 1 h–30′ UCN-01 30′–20′ caffeine 10 mM 30′ caffeine 5 mM fork to fork 30′–30′ caffeine 5 mM fork to fork 1 h–30′ 300 nM UCN-01 fork to fork 30′–20′ caffeine 5 mM. Fork length BrdU 30′–20′ caffeine 10 mM. Fork length BrdU 30′ caffeine 30′–30′ caffeine UCN-01 30′ caffeine 30′–30′ caffeine Figure 7 Chk1 regulates replication origin density in primary avian fibroblasts. Replication structures of chick embryo fibroblasts were visualised using the procedures described in Figure 2. Replicon size (A–C) was measured using the separation of sister forks (panel A) after labelling with biotin-dU and structures in untreated cells compared with those in cells treated with caffeine or UCN-01. BrdU labelling was also used to determine the rate of fork elongation (panel B) under the same conditions. Parameters defining average replicon structure under different conditions are shown (panel C). The number of active replication factories (D, E) and organisation of active replication foci (F) in cells treated with caffeine and UCN-01 were compared with untreated cells. Changes in the number of active DNA foci in treated cells were shown to 0 0 0 be statistically significant using Mann–Whitney test: 30 caffeine versus control (Po0.05; U-value 78); 30 –30 caffeine versus control (Po0.005; U-value 88); UCN-01 versus control (Po0.02; U-value 82). Scale bars (5 and 0.5mm in Detail) are shown on individual images. maintains replication fork stability and blocks initiation of (Miao et al, 2003; Syljuasen et al, 2005), consistent with Chk1 latent replication origins (Kastan and Bartek, 2004; Sancar contributing to the regulation of DNA synthesis. In support of et al, 2004). During the damage response, activation of Chk1 this idea, recent studies from embryonic systems have sug- leads to phosphorylation of Cdc25, which prevents down- gested that the ATR/Chk1-dependent regulation of origin stream activation of the CDK/cyclin complexes that are function might define the sequential activation of replication required to activate latent origins. By maintaining the phy- origins during S phase (discussed in Fisher and Mechali, siological balance of this pathway, Chk1 is also thought to 2004; Shechter and Gautier, 2005). However, a global increase contribute to the regulation of DNA synthesis in the absence in DNA synthesis in cells with reduced Chk1 activity conflicts of replicative stress (Zhao et al, 2002; Sorensen et al, 2003). with our recent observation that loss of Chk1 function The importance of balance within this regulatory pathway is reduces the rate of replication fork progression to B50% of þ / emphasised by the phenotype of Chk1 cells from trans- that in wt cells (Petermann et al, 2006). In order to resolve genic mice (Lam et al, 2004). Such cells maintain three clear this conflict, we have used quantitative analysis of nascent haploinsufficiency phenotypes: inappropriate S-phase entry, replicons in spread DNA fibres. Using both transformed and accumulation of DNA damage during replication and failure primary cells of human and avian origin, this approach to restrain mitotic entry. Inappropriate cell cycle transitions in provides the first formal proof that the density of active these cells correlate with maintenance of high levels of replication origins is increased by at least 2- to 3-fold when Cdc25A. Chk1 activity is compromised (Figures 1, 2, 4 and 7). In somatic mammalian cells, loss of Chk1 function corre- Consistent with a role for Chk1 in modulating the density lates with a slight (20–30%) increase in global replication of active replication origins during normal S phase, our 2728 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al analysis of DNA fibres showed that inhibition of Chk1 ride Chk1-dependent suppression of late replicons in an compromises the efficacy of DNA replication and leads to early-S-phase cell (Shechter et al, 2004), the late origins of reduced rates of elongation (Figures 2 and 7). Compromised somatic human cells are not activated because they are replication at stalling forks correlates with an accumulation unable to access active replication machinery. This implies of aberrant DNA structures and activation of secondary, that the temporal programme of replication factory assembly previously latent, origins (Figures 3 and 6) within the vicinity plays a dominant role in defining which potential origins can of the damaged forks. This activation of normally suppressed be activated at different times of S phase. origins in cells with compromised Chk1 activity accounts for In avian cells in contrast, loss of Chk1 function correlated the observed increase in density of active origins, and implies with a significant increase in the number of active replication that somatic vertebrate cells have an innate mechanism that foci and some disorganisation of the temporal programme of balances the rate of elongation at replication forks with the S-phase synthesis (Figures 4 and 7). This difference between density of active replication origins. human and avian cells raises interesting questions concern- Alterations in the balance of the normal regulatory ma- ing the evolution of S-phase regulation. ATR and Chk1 play a chinery appear to account—at least in part—for our observa- key role in local (within individual replication factories) tions. It is known that reduced expression or inhibition of regulation of origin activation in early stages of amphibian Chk1 leads to accumulation of Cdc25A, which in principle (Xenopus) development and in avian and mammalian so- would support super-activation of replication origins matic cells. However, in the in vitro Xenopus system, (Sorensen et al, 2003; Lam et al, 2004). We confirm that the temporal activation of replicon clusters is lost if ATR is Cdc25A concentration increases by B5-fold in wt DT40 cell inhibited (Marheineke and Hyrien, 2004). In human cells, treated with caffeine or UCN-01 (Supplementary Figure S3), in contrast, S-phase timing is preserved when ATR function and that the origin associated protein Cdc45 is increased by is compromised (Figures 1 and 2). Intriguingly, somatic about two-fold in human fibroblasts (Supplementary Figure S1) avian cells seem to have evolved a hybrid organisation and DT40 (Supplementary Figure S3) when Chk1 activity is in which the timing programme is disordered (Figures 4 inhibited. and 7) but the duration of S phase is maintained (Zachos et al, 2005). These differences presumably imply that the Does Chk1 contribute to S-phase progression in somatic regulatory pathways that control S-phase progression are able vertebrate cells? to interact (i.e. complement) to different extents in mamma- lian and avian cells. This conclusion is not entirely un- In nuclei assembled using Xenopus egg extract, ATR/Chk1 founded, as deletion of Chk1 in the mouse is known to be regulate the sequential activation of early and late replication cell lethal (Liu et al, 2000) whereas avian DT40 Chk1 cells origins (Marheineke and Hyrien, 2004; Shechter et al, 2004). are viable. It is important to recognise, however, that this embryonic system is very different from somatic cells in key respects. First, assembled nuclei have simple chromatin architecture and no transcription and second, the cell cycle is very rapid, A model for Chk1 function during normal S phase with an S phase of B30 min and correspondingly short Experiments present here support the idea that Chk1 con- replicons of B15 kb (Blow et al, 2001). Although replicon tributes to the efficacy of replication at active replication clusters are activated throughout this short S phase (Blow forks and couples defects in synthesis at active forks to the et al, 2001), it is unknown if this is of physiological signifi- local activation of nearby latent origins. This implies that cance or reflects the stochastic nature of replication in this even during a normal S phase, a Chk1-dependent system system. In clear contrast, somatic mammalian cells have been continually senses the status of active replicons within repli- shown to have well-established S-phase programmes, with cation factories and monitors the number of active forks to specific regions of the genome replicated at precise times ensure progress of the synthetic process. (Jackson and Pombo, 1998; Sadoni et al, 2004). Progress An important feature of this model is the provision of through the programme can be correlated with the spatial latent origins, which are suppressed during normal synthesis architecture of the active replication sites (Jackson, 1995) and but serve to rescue replication if for any reason the active concomitantly correlated with the assembly of different forks might fail. In fact, it has been known for many years classes of chromatin at different times during S phase that immediately before S phase, replicons have B10-fold (Zhang et al, 2002). excess of potential replication origins—defined by MCM2–7 Despite these clear differences, it is interesting to compare complexes—which are clustered into licensing groups (dis- our studies with in vitro observations in Xenopus extracts cussed in Woodward et al, 2006). The redundant MCM (Marheineke and Hyrien, 2004; Shechter et al, 2004), which complexes are displaced from chromatin during replication, suggested that ATR and Chk1 regulate S-phase progression by but also appear to provide a rescue function if active forks are suppressing the activation of late origins during early S phase aborted because of DNA damage (Woodward et al, 2006). (Shechter et al, 2004). In human cells, we show that a This means that under normal conditions, only a minority of mechanism that involves Chk1 suppresses the majority of potential origins is actually used, and that a mechanism must potential origins that exist in active replicons; potential operate to limit origin activation so that once the required origins are known to outnumber those that are used by at number of origins has been activated the use of proximal least 10-fold (discussed in Woodward et al, 2006). When potential origins is suppressed. The data presented here Chk1 activity was inhibited, super-activation of latent origins support the idea (Shechter and Gautier, 2005) that the ATR/ was restricted to regions of the genome that were already Chk1 pathway controls the activation and suppression of engaged with active replication factories (Figures 1H and 2I). replication origins to regulate the density of active origins Hence, while caffeine treatment might be expected to over- throughout S phase of somatic vertebrate cells. &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2729 | | Chk1 regulates origin density A Maya-Mendoza et al 10 min at 01C. Coverslips were then rinsed in PBS and incubated in Materials and methods fresh medium for 30 min. MRC5 and CEF cells were transfected directly with 30ml of transfection mix in 35 mm Petri dishes and Cell culture incubated on ice for 10 min, rinsed with cold PBS and incubated in HeLa cells were grown in DMEM (Sigma) with 5% FBS and fresh medium for 30 min. antibiotics. DT40 cells were grown in RPMI-1640 (Sigma) with 10% These loading conditions use very low precursor concentrations FBS, 1% chicken serum, 10 M b-mercaptoethanol and antibiotics. that were optimised to transiently support normal rates of fork Early-passage MRC5 cells (ATCC cell line CCL-171—normal human elongation. The treatment has no short- or long-term effects on diploid fibroblasts from lung) were grown in MEM, L-glutamine, cell cycle duration and cell viability. For example, replication sodium pyruvate, non-essential amino acids with 10% FBS forks in DT40 cells analysed 10 min after loading travelled and antibiotics. CEF (Kilbey et al, 1996) were grown in DMEM, 12.1972.61 kb (n¼ 52), which corresponds with the average L-glutamine, non-essential amino acids, sodium pyruvate, 1% of elongation rate of 1.2 kb/min in these cells (Petermann et al, chicken serum, 10% FBS and antibiotics. 2006). In addition, the Chk1-dependent DNA damage checkpoint was not activated by these loading and replication conditions DNA fibre experiments (Supplementary Figure S4). Replication tracks were labelled in culture medium containing 25mM BrdU. For dual labelling, cultures were pulse labelled with Immunofluorescence and direct labelling of DNA foci 25mM BrdU, washed and then labelled with 250mM iodo- / / 5 wt, Chk1 and Chk2 DT40 cells at 510 cells/ml were deoxyuridine (IdU). These conditions are known to support natural labelled with 25mM BrdU, 250mM IdU or transfected with the rates of fork elongation (Petermann et al, 2006). Cells were labelled specific dUTP analogue. Cells were washed 3 in cold PBS and with modified dUTPs as detailed below. UCN-01 was used at 300 nm 110 cells swollen in 0.075 M KCl for 15 min at 371C (Jackson and and caffeine at 5 mM. Pombo, 1998). Nuclei were fixed with methanol/acetic acid (3:1), DNA fibre spreads were prepared as previously described added dropwise onto washed microscope slides, and dried. HCl (Jackson and Pombo, 1998). BrdU-labelled tracks were detected denaturation and immunolabelling were performed as detailed with BrdU anti-sheep antibody (Biodesign; M20105S; 1:1000 before (Jackson and Pombo, 1998). dilution; 1 h at 201C) using either Cy3- or AlexaFluor-488- For double labelling experiments, DT40 nuclei fixed with conjugated donkey anti-sheep secondary antibody. Biotin-11-dUTP methanol/acetic acid or HeLa, MRC5 and CEF cells fixed in 4% detection was carried out using a mouse monoclonal antibody PF were rinsed three times in PBS, two times in ddH O and then (Clone BN-34; Sigma; 1:1000 dilution; 1 h at 201C) and Dig-11-dUTP 2 incubated with HCl. The slides were rinsed in PBS and PBSþ , (Roche) detection using a sheep polyclonal antibody (Roche; 1:1000 blocked and incubated with rat anti-BrdU (Immunologicals Direct dilution; 0.5 h at 201C) and appropriate Cy3- or AlexaFluor-488- Clone BU 1/75; 1:1000 dilution; 1 h at 201C), washed 3 in PBS conjugated second antibody. and 3 in PBSþ and AlexaFluor-488-conjugated donkey anti-rat Fibres were examined using a Zeiss LSM 510 confocal micro- antibody (1:1000 dilution; 1 h; 41C). The slides were washed 3 in scope using a  100 (1.4 NA) lens, labelled tracks measured using PBS and 3 in PBSþ , and IdU detected using BrdU anti-sheep the LSM software (white bars on individual images show examples antibody (1:1000 dilution; 15 h at 41C). Slides were then washed of measurements recorded) and converted to kilobase pairs using a 3 in PBS, 3 in PBSþ and incubated with Cy3-conjugated conversion factor of 1mm¼ 2.59 kb (Jackson and Pombo, 1998). donkey anti-sheep antibody (1:1000 dilution; 1 h at 41C). Finally, Measurements were recorded in randomly selected fields (selected the slides were washed 3 in PBSþ,3 in PBS, incubated with at low power) from dispersed, untangled areas of the DNA spread. 5mg/ml Hoechst 33258 (Sigma) for 10 min, rinsed 3 in PBS and As the analysis of single, unbroken fibres is a key feature of this mounted with 50:50 PBS–glycerol. study, routine quality control for spreading of different cell types For nuclei labelled with Cy3-dUTP and/or AlexaFluor-488- under different experimental conditions was performed using direct conjugated dUTP, slides were washed 3 in PBS, incubated with DNA labelling with YOYO (Merrick et al, 2004) or BrdU 5mg/ml Hoechst 33258 (Sigma) for 10 min, and then rinsed 3  in immunolabelling of fibres from cells labelled with BrdU for B24 h. PBS and mounted with PBS–glycerol. Slides were examined using a Zeiss LSM 510META confocal Replication foci in situ microscope using a  100(1.4 NA) lens. 3D images were generated Modified nucleoside triphosphates were introduced into cells using TM using Z stacks and processed in Imaris software. FuGene (Roche). Briefly, DT40 cells were pelleted (200 g, 4 min), washed 3  in cold PBS and 110 cells in 10ml PBS transfected by incubating with transfection mix (12ml PBS, 3ml of FuGene and 1ml Supplementary data (1 nmol) of the required dUTP analogue) for 10 min at 01C. After Supplementary data are available at The EMBO Journal Online transfection, the cells were washed in fresh medium (0.5 ml of (http://www.embojournal.org). medium, 200 g, 4 min) and incubated in medium (0.5 ml) for 30 min at 371C. For double labelling, the same protocol was used for the second dUTP analogue. For experiments using BrdU, cultures were Acknowledgements pulse labelled with 25mM BrdU either before or after transfection. HeLa cells were grown on microscope coverslips and transfected This work was funded by the BBSRC (linked project grant BBS/B/ with biotin-11-dUTP. Coverslips were rinsed in PBS and placed over 06091 to DAJ and KWC). We thank Chi Tang and other colleagues a 7.5ml droplet of transfection mix on parafilm and incubated for for technical advice and support. References Berezney R, Dubey DD, Huberman JA (2000) Heterogeneity of Jackson DA, Pombo A (1998) Replicon clusters are stable units of eukaryotic replicons, replicon clusters, and replication foci. chromosome structure: evidence that nuclear organization con- Chromosoma 108: 471–484 tributes to the efficient activation and propagation of S phase in Blow JJ, Gillespie PJ, Francis D, Jackson DA (2001) Replication human cells. J Cell Biol 140: 1285–1295 origins in Xenopus egg extracts are 5–15 kb apart and are acti- Kastan MB, Bartek J (2004) Cell-cycle checkpoints and cancer. vated in clusters that fire at different times. J Cell Biol 152: 15–26 Nature 432: 316–323 Fisher D, Mechali M (2004) Sleeping policemen for DNA replica- Kilbey A, Black EJ, Unlu M, Gillespie DAF (1996) The v-Jun tion? Nat Cell Biol 6: 576–577 oncoprotein replaces p39 c-Jun as the predominant AP-1 consti- Gilbert DM (2002) Replication timing and transcriptional control: tuent in ASV17-transformed fibroblasts: implications for SAPK/ beyond cause and effect. Curr Opin Cell Biol 14: 377–383 JNK-mediated signal transduction. Oncogene 12: 2409–2418 Jackson DA (1995) Nuclear organization—uniting replication foci, Lam H, Liu Q, Elledge SJ, Rosen JM (2004) Chk1 is haploinsufficient chromatin domains and chromosome structure. Bioessays 17: for multiple functions critical to tumor suppression. Cancer Cell 6: 587–591 45–59 2730 The EMBO Journal VOL 26 NO 11 2007 &2007 European Molecular Biology Organization | | Chk1 regulates origin density A Maya-Mendoza et al Liu Q, Guntuku S, Cui XS, Matsuoka S, Cortez D, Tamai K, Luo G, Shechter D, Gautier J (2005) ATM and ATR check in on origins—a Carattini-Rivera S, DeMayo F, Bradley A, Donehower LA, Elledge dynamic model for origin selection and activation. Cell Cycle 4: SJ (2000) Chk1 is an essential kinase that is regulated by Atr and 235–238 required for the G(2)/M DNA damage checkpoint. Genes Dev 14: Sorensen CS, Syljuasen RG, Falck J, Schroeder T, Ronnstrand L, 1448–1459 Khanna KK, Bartek J, Lukas J (2003) Chk1 regulates the S phase Machida YJ, Hamlin JL, Dutta A (2005) Right place, right time, and checkpoint by coupling the physiological turnover and ionizing only once: replication initiation in metazoans. Cell 123: 13–24 radiation-induced accelerated proteolysis of Cdc25A. Cancer Cell Miao H, Seiler JA, Burhans WC (2003) Regulation of cellular and 3: 247–258 SV40 virus origins of replication by Chk1-dependent intrinsic and Syljuasen RG, Sorensen CS, Hansen LT, Fugger K, Lundin C, UVC radiation-induced checkpoints. J Biol Chem 278: 4295–4304 Johansson F, Helleday T, Sehested M, Lukas J, Bartek J (2005) Marheineke K, Hyrien O (2004) Control of replication origin Inhibition of human Chk1 causes increased initiation of DNA density and firing time in Xenopus egg extracts—role of a replication, phosphorylation of ATR targets, and DNA breakage. caffeine-sensitive, ATR-dependent checkpoint. J Biol Chem 279: Mol Cell Biol 25: 3553–3562 28071–28081 Takai H, Tominaga K, Motoyama N, Minamishima YA, Nagahama Merrick CJ, Jackson D, Diffley JFX (2004) Visualization of altered H, Tsukiyama T, Ikeda K, Nakayama K, Nakanishi M, Nakayama replication dynamics after DNA damage in human cells. J Biol K (2000) Aberrant cell cycle checkpoint function and early Chem 279: 20067–20075 embryonic death in Chk1(/) mice. Genes Dev 14: 1439–1447 Norio P, Kosiyatrakul S, Yang QX, Guan ZQ, Brown NM, Thomas S, Woodward AM, Go¨hler T, Luciani MG, Oehlmann M, Ge X, Gartner Riblet R, Schildkraut CL (2005) Progressive activation of DNA A, Jackson DA, Blow JJ (2006) Excess Mcm2–7 license dormant replication initiation in large domains of the immunoglobulin origins of replication that can be used under conditions of heavy chain locus during B cell development. Mol Cell 20: replicative stress. J Cell Biol 173: 673–683 575–587 Zachos G, Rainey MD, Gillespie DAF (2003) Chk1-deficient tumour Petermann E, Maya-Mendoza A, Zachos G, Gillespie DAF, Jackson cells are viable but exhibit multiple checkpoint and survival DA, Caldecott KW (2006) Chk1 requirement for high global rates defects. EMBO J 22: 713–723 of replication fork progression during normal vertebrate S phase. Zachos G, Rainey MD, Gillespie DAF (2005) Chk1-dependent S–M Mol Cell Biol 26: 3319–3326 checkpoint delay in vertebrate cells is linked to maintenance of Sadoni N, Cardoso MC, Stelzer EHK, Leonhardt H, Zink D (2004) viable replication structures. Mol Cell Biol 25: 563–574 Stable chromosomal units determine the spatial and temporal Zhao H, Watkins JL, Piwnica-Worms H (2002) Disruption of the organization of DNA replication. J Cell Sci 117: 5353–5365 checkpoint kinase 1/cell division cycle 25A pathway abrogates Sancar A, Lindsey-Boltz LA, Unsal-Kacmaz K, Linn S (2004) ionizing radiation-induced S and G2 checkpoints. Proc Natl Acad Molecular mechanisms of mammalian DNA repair and the DNA Sci USA 99: 14795–14800 damage checkpoints. Annu Rev Biochem 73: 39–85 Zhang JM, Xu F, Hashimshony T, Keshet N, Cedar H (2002) Shechter D, Costanzo V, Gautier J (2004) ATR and ATM regulate the Establishment of transcriptional competence in early and late S timing of DNA replication origin firing. Nat Cell Biol 6: 648–655 phase. Nature 420: 198–202 &2007 European Molecular Biology Organization The EMBO Journal VOL 26 NO 11 2007 2731 | |

Journal

The EMBO Journal – Springer Journals

Published: Jun 6, 2007

Keywords: checkpoint proteins; DNA foci; DNA replication; replication origins; S‐phase programme

There are no references for this article.