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Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos JCB: ARTICLE Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos 1 1 2 1 Antonia H. Holway, Seung-Hwan Kim, Adriana La Volpe, and W. Matthew Michael The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Istituto di Genetica e Biofi sica “Adriano Buzzati-Traverso,” Consiglio Nazionale delle Ricerche, 80125 Naples, Italy n most cells, the DNA damage checkpoint delays but not in the germ line. Silencing requires rad-2, gei-17, cell division when replication is stalled by DNA and the polh-1 translesion DNA polymerase, which I damage. In early Caenorhabditis elegans embryos, suppress replication fork stalling and thereby eliminate however, the checkpoint responds to developmental the checkpoint-activating signal. These results explain signals that control the timing of cell division, and how checkpoint activation is restricted to developmen- checkpoint activation by nondevelopmental inputs dis- tal signals during embryogenesis and insulated from rupts cell cycle timing and causes embryonic lethality. DNA damage. They also show that checkpoint activa- Given this sensitivity to inappropriate checkpoint acti- tion is not an obligatory response to DNA damage and vation, we were interested in how embryos respond to that pathways exist to bypass the checkpoint when DNA damage. We demonstrate that the checkpoint re- survival depends on uninterrupted progression through sponse to DNA damage is actively silenced in embryos the cell cycle. Introduction Early embryogenesis in many organisms, including Xenopus In embryonic cells, the ATR checkpoint is activated by laevis, Drosophila melanogaster, and Caenorhabditis elegans, endogenous, developmentally programmed cues. The nature of is characterized by rapid progression through the cell cycle (for these signals is not defi ned, but it is clear that developmental review see O’Farrell et al., 2004). Features of early embryonic activation of the checkpoint is important for regulating the tim- cell cycles that distinguish them from somatic cycles include ing of cell division during early embryogenesis. Two examples cell division in the absence of cell growth and a lack of Gap highlight this importance. In D. melanogaster, the mei-41 (ATR) phases. Another important difference between somatic and and grapes (Chk1) genes affect a developmentally programmed embryonic cell cycles concerns the utilization of S phase check- slowing of the cell cycle that occurs at the midblastula transition point pathways. In somatic cells, the S phase checkpoint senses (Sibon et al., 1997, 1999; Su et al., 1999; Yu et al., 2000). Fly DNA damage and responds by delaying progression into mito- embryos perform 13 rounds of rapid and synchronous cell divi- sis (for reviews see Bartek et al., 2004; Sancar et al., 2004). sion before the midblastula transition. After cycle 13, the mei41/ The protein kinases ATR and Chk1 are central to S phase check- grapes checkpoint is activated by an endogenous signal, and point signaling. DNA damage causes replication fork stalling, this slows the cell cycle down. Slowing of the cell cycle in turn which in turn activates ATR and promotes the ATR-directed allows for zygotic transcription to begin, and the control of cell phosphorylation of Chk1. Activated Chk1 delays cell cycle pro- division is thereby transferred from maternal to zygotic regulators. gression through attenuation of core cell cycle regulators such as In mei-41 or grapes mutants, the cell cycle does not slow the Cdc25 protein phosphatase. Thus, in somatic cells, a major down, zygotic control of the cell cycle does not happen on function of the ATR checkpoint is to delay cell cycle progression schedule, and the embryo dies. Therefore, in D. melanogaster, in response to DNA damage until replication can fi nish. the checkpoint plays an important role in remodeling the cell cycle so that zygotic transcription can begin on schedule. Another example of DNA damage–independent utiliza- tion of the ATR checkpoint is found in C. elegans. The one-cell Correspondence to W Correspondence to W.. Matthew Michael: [email protected] Matthew Michael: [email protected] vard.edu embryo, or P0 cell, divides asymmetrically to produce the Abbreviations used in this paper: HU, hydroxyurea; MMS, methanesulphonate; Abbreviations used in this paper: HU, hydroxyurea; MMS, methanesulphonate; smaller (P1) and the larger (AB) daughter cells. The next round PCNA, proliferating cell nuclear antigen; RNAi, RNA inter PCNA, proliferating cell nuclear antigen; RNAi, RNA interference. ference. The online version of this ar The online version of this article contains supplemental material. ticle contains supplemental material. of cell division is asynchronous: AB divides fi rst, followed by © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 172, No. 7, March 27, 2006 999–1008 http://www.jcb.org/cgi/doi/10.1083/jcb.200512136 JCB 999 THE JOURNAL OF CELL BIOLOGY P1 about 2 min later. This 2-min delay is controlled in part through differential activation of the S phase checkpoint in the P1 cell (Brauchle et al., 2003). Developmental checkpoint acti- vation in the early embryo requires the C. elegans homologues of ATR (atl-1) and Chk1 (chk-1). Checkpoint-mediated asyn- chrony in cell division is extremely important to embry- onic patterning in C. elegans. When asynchrony is reduced, through loss of chk-1, the germ line fails to develop and the ani- mal is sterilized (Brauchle et al., 2003; Kalogeropoulos et al., 2004). Extending the asynchrony also has deleterious conse- quences. Hypomorphic mutations in div-1, a gene encoding DNA polymerase α, cause replication problems that result in inappropriate activation of the chk-1 pathway (Encalada et al., 2000; Brauchle et al., 2003). The div-1–mediated activation of chk-1 extends the asynchrony in cell division, and this results in mislocalization of developmental regulators, embryonic patterning defects, and lethality (Encalada et al., 2000). From these examples it is clear that, although checkpoint activation is important for development, it must only occur in response to developmental signals and not in response to un- scheduled events such as replication problems. A common source of replication problems in wild-type cells is DNA damage, and thus it would seem that early embryogenesis in C. elegans would be particularly sensitive to DNA damage be- cause of the deleterious consequences of unscheduled check- point activation. Paradoxically, this is not so, as previous work has shown that wild-type embryos are resistant to relatively high amounts of both UV light and the alkylating agent methyl methanesulphonate (MMS; Hartman and Herman, 1982; Holway et al., 2005), two DNA-damaging agents that are known to cause replication problems and subsequent check- point activation (Lupardus et al., 2002; Stokes et al., 2002; Tercero et al., 2003). We resolve this paradox by showing that the checkpoint is actively silenced during the DNA damage response in early embryos. We go on to defi ne genetic require- ments and the basis for checkpoint silencing. Our results identify a novel developmental mechanism that ensures that Figure 1. Differential checkpoint responses to DNA damage in the germ line cell cycle progression is not attenuated by DNA damage, thus and early embryo. (A–C) Gonads were dissected from adult hermaphrodites, providing embryos with a chance of survival even when their fi xed, and stained with Hoechst 33258 to visualize the nuclei. Where indicated, the worms had been exposed to MMS (B and C; 0.005% MMS) chromosomes are heavily damaged. or atl-1 RNAi (C) before fi xation. Nuclei were then visualized and photographed by fl uorescence microscopy. (D) Schematic depiction of the fi rst cell cycle during C. elegans embryogenesis. (E) Wild-type (N2) em- Results bryos were cultured on regular media (control), media containing 75 mM HU (HU), media containing HU and E. coli expressing double-stranded Levels of DNA-damaging agents that RNA against chk-1 (+ HU + chk-1 RNAi), or media containing 0.005% trigger a checkpoint arrest in germ cells MMS (+ MMS) or were exposed to 100 J/m of UV light (+ UV). The tim- ing of the fi rst embryonic cell cycle was then determined by microscopic do not activate the checkpoint in embryos examination of living embryos, and the mean time, from a minimum of It was not known whether the C. elegans checkpoint pathway 10 samples per data point, for P0 S phase progression is displayed. can sense the types of DNA damage that cause replication stress, P0 S phase progression is defi ned as the elapsed time required to progress from step ii to v in the diagram in D. Error bars represent one standard such as alkylation or UV light–induced damage. Previous work deviation from the mean. (F) Same as E, except that the media contained has shown that nuclei in the mitotic zone of the hermaphrodite the indicated concentrations of MMS. gonad, a nonembryonic tissue, undergo checkpoint-dependent cell cycle arrest in response to replication blocks and ionizing radiation (Gartner et al., 2000; MacQueen and Villeneuve, Hoechst 33258 to visualize nuclei. MMS reduced the number 2001). This arrest is refl ected by a reduction in nuclei number of nuclei within the mitotic zone from a mean of 35 to a mean and an increase in nuclear size. To see whether MMS and/or UV of 22 (Fig. 1, A and B; and Table I). The effect of MMS in light induced checkpoint activation in the gonad, animals were the germ line was reversed when the checkpoint gene atl-1, exposed to 0.005% MMS and then fi xed and stained with the worm orthologue of ATR (Brauchle et al., 2003), was 1000 JCB • VOLUME 172 • NUMBER 7 • 2006 Table I. Nuclei in the mitotic zone of the hermaphrodite gonad Table II. Embryonic lethality after chronic MMS exposure Genotype Condition Nuclei count MMS Time Emb %h % Wild type Control 35 ± 2.6 —16 0 Wild type MMS 22.5 ± 2.0 —32 0 Wild type UV 20.9 ± 3.5 0.001 16 0 atl-1 RNAi Control 34.1 ± 2.9 0.001 32 0 atl-1 RNAi MMS 33.4 ± 4.0 0.005 16 1.3 atl-1 RNAi UV 34.8 ± 4.3 0.005 32 2.4 gei-17 RNAi Control 34.6 ± 3.9 0.01 16 73 gei-17 RNAi MMS 22.6 ± 3.1 0.01 32 94 gei-17 RNAi UV 0.025 16 96 21.0 ± 2.4 0.025 32 98 Young adult hermaphrodites of the indicated genotype were fi xed and stained with Hoechst 33258 to visualize nuclei in the mitotic zone of the gonad. The Young adult hermaphrodites were transferred to media containing the indicated nuclei within a fi xed volume were then counted for a minimum of 10 samples per concentration of MMS. After 16 h, the animals were transferred to fresh MMS data point. Shown are these counts with the standard deviation. Condition refers plates, and incubation was continued for an additional 16 h. At the end of to animals that were not exposed to DNA-damaging agents (control), animals each 16-h incubation, the eggs that had been laid were counted and then that were exposed to 0.005% MMS (MMS), or animals that were exposed to counted again 20 h later. Emb refers to the percentage of embryonic lethality 100 J/m of UV light (UV). or the percentage of eggs that failed to hatch during the 20 h after removal of the adults. depleted by RNA interference (RNAi; Fig. 1 C and Table I). Similar results were obtained when animals were irradiated of embryos to avoid a checkpoint response to MMS is saturable. with 100 J/m of UV light (Table I). We conclude that germ They also indicate that checkpoint silencing and survival of DNA cells undergo checkpoint-dependent cell cycle arrest upon damage are linked, and this is consistent with previous work exposure to either MMS or UV light and that, therefore, the showing that even modest perturbations in the timing of cell divi- C. elegans checkpoint can indeed sense MMS- and UV light– sion are lethal to the developing embryo (Encalada et al., 2000). induced damage. The effect of MMS and UV light on cell cycle progression Embryonic checkpoint silencing in the early embryo was examined next. Fig. 1 D shows the is independent of lesion repair major events of the fi rst mitotic interphase in the early embryo. yet under genetic control After fertilization (step i), the female pronucleus migrates across A simple explanation for checkpoint silencing is that embryos the embryo, or P0 cell, where it meets and apposes the male rapidly repair damaged DNA. However, extensive analysis of pronucleus (steps ii–iv). DNA replication then fi nishes, and mi- the kinetics of DNA repair in C. elegans has been reported tosis is initiated by nuclear envelope breakdown (step v). Previ- (Hartman and Herman, 1982; Hartman, 1984; Hartman et al., ous work has shown that replication stress–induced checkpoint 1989; Jones and Hartman, 1996), and these studies demonstrate activation, as triggered by the replication inhibitor hydroxyurea that repair is unlikely to account for checkpoint silencing in the (HU), occurs at the one-cell stage (Brauchle et al., 2003). This embryo. For example, >80% of (6-4) photoproducts remain in checkpoint requires the chk-1 gene (Brauchle et al., 2003) and embryos 3 h after a dose of 50 J/m of UV light is delivered prevents the transition from step iv to v in Fig. 1 D. Animals (Hartman et al., 1989). The data in Fig. 1 E were collected 1 h were exposed to MMS, UV light, or, as a positive control, HU, after a dose of 100 J/m was delivered, and thus the embryo and the timing of the fi rst cell cycle was determined by direct could not possibly have repaired even a modest percentage of microscopic visualization of living embryos. As shown in Fig. 1 E, the damage in that short a period of time. We conclude that cell and as previously reported (Brauchle et al., 2003), when em- cycle progression occurs unimpeded even when the level of bryos were treated with HU, there was a signifi cant delay in damage present greatly exceeds the capacity of the embryo to progression through the P0 cell cycle. This delay was check- repair it. point dependent, as it was reversed after depletion of chk-1 by If embryonic cell cycle progression is truly independent RNAi. In contrast to HU, we did not detect a signifi cant P0 cell of repair, then mutant embryos that are defi cient in DNA repair cycle delay when the embryos were exposed to MMS or UV would nonetheless exhibit normal cell cycles after DNA damage. light (Fig. 1 E). We conclude that the amounts of MMS or UV To test this, we examined cell cycle progression in early rad-3 light that are suf cient to acti fi vate a checkpoint response in the embryos. rad-3 mutant embryos have a defect in excision repair germ line (0.005% and 100 J/m , respectively) cannot do so in and are consequently very sensitive to both MMS (Fig. 2 A) and early embryos. We refer to this phenomenon as early embryonic UV light (Hartman and Herman, 1982; Hartman et al., 1989). checkpoint silencing. The rate of repair in rad-3 embryos has been determined and is We next tested how much MMS embryos could endure threefold lower than wild type (Hartman et al., 1989). Despite before a delay in cell division was detected. For this, we timed this reduced capacity for repair, however, the timing of cell the fi rst cell division after exposure to a range of MMS concen- division in rad-3 mutant embryos was indistinguishable from trations and found that concentrations >0.005% caused both a wild type after exposure to either UV light or MMS (Fig. 2 B). delay in progression through S phase (Fig. 1 F) and high levels The dose of UV light used in the experiment is suffi cient to of embryonic lethality (Table II). These data show that the ability kill 100% of the rad-3 mutant embryos and <10% of wild-type DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1001 indistinguishable from wild type (Hartman, 1984). Thus repair- defi cient rad-3 mutants have normal cell cycles after DNA damage, whereas repair-profi cient rad-2 mutants do not. This shows that a process that is independent of DNA repair is responsible for preventing checkpoint activation during the early embryonic cell cycle. Consistent with this, we also found that RNAi-mediated depletion of another excision repair gene, the C. elegans homologue of the human XPF endonuclease (F10G8.7), renders embryos extremely sensitive to both UV light (Fig. S1, avail- able at http://www.jcb.org/cgi/content/full/jcb.200512136) and MMS (Fig. 2 A) yet had no affect on cell cycle progression in the early embryo (Fig. 2 B). We conclude that checkpoint silencing is independent of lesion repair (based on the results with rad-3 and F10G8.7) yet nonetheless under genetic control (based on the results with rad-2). gei-17 suppresses the checkpoint response to DNA damage but not developmental signals or stalled replication forks in early embryos The fi nding that checkpoint silencing is under genetic control prompted a search for genes that silence the checkpoint when DNA damage is present. The rad-2 gene has not yet been cloned, and we are currently working toward accomplishing this. Recent work from our laboratory has shown that the gei-17 gene, which encodes an E3 SUMO ligase related to yeast SIZ1 and human PIAS1, is an important participant in the embryonic DNA damage response in C. elegans (Holway et al., 2005). Depletion of gei-17 by RNAi renders embryos sensitive to both MMS (Holway et al., 2005) and UV light (Fig. S1). MMS- exposed gei-17 RNAi early embryos display abnormal nuclear morphology, characterized by fused nuclei and anaphase bridg- ing (Holway et al., 2005). These results suggested that gei-17 is important for early embryonic cell cycle progression when dam- age is present and prompted us to examine the kinetics of cell division in gei-17 RNAi early embryos. MMS exposure delayed progression through the P0 S phase in gei-17 RNAi embryos. At 0.005% MMS, we observed an 440-s delay (Fig. 3 A) and at 0.001% MMS the delay was 300 s (not depicted). These data Figure 2. Normal progression through the early cell cycles in excision demonstrate that S phase takes longer in gei-17 RNAi embryos repair–defi cient embryos exposed to DNA damage. (A) MMS sensitivity exposed to 0.001% MMS than it does in wild-type embryos of embryos of the indicated genotype. Details on the MMS sensitivity assay can be found in Materials and methods and in Holway et al. (2005). exposed to 10-fold more MMS (the wild-type delay at 0.01% (B) Timing of the P0 S phase was determined as in Fig. 1 E for embryos of MMS was 120 s; Fig. 1 F). To determine whether these MMS- the indicated genotype. Control refers to regular media, + MMS refers to induced effects were caused by activation of the checkpoint, we media containing 0.005% MMS, and + UV refers to exposure to 100 J/m of UV light. codepleted gei-17 with chk-1. As was the case with rad-2, code- pletion of gei-17 with chk-1 reversed the MMS-induced delay in progression through the P0 cell cycle (Fig. 3 A). This result embryos (unpublished data; see Hartman and Herman [1982] demonstrates that gei-17 activity suppresses checkpoint activa- for UV sensitivity of rad-3 mutants). Interestingly, another tion in response to DNA damage in the early embryo. radiation- and MMS-sensitive mutant, rad-2, showed altered To see whether the effect of gei-17 on checkpoint activa- progression through the fi rst cell cycle after exposure to UV or tion was specifi c for DNA damage, we next examined check- MMS (Fig. 2, A and B). rad-2 mutant embryos delayed progres- point activation in gei-17 RNAi embryos in response to both sion through the P0 S phase in a manner that was dependent on HU and developmental signals. For the HU experiment, we DNA damage and similar to wild-type embryos exposed to HU. used a lower concentration of HU than that used in Fig. 1 The damage-induced delay in rad-2 embryos was checkpoint (25 as opposed to 75 mM), and this resulted in a more modest dependent, as it was reversed when chk-1 was depleted by RNAi delay in cell division in wild-type embryos (160 s delay after (Fig. 2 B). Importantly, the rate of repair in rad-2 mutants is 25 mM HU in contrast to the 475-s delay after 75 mM; Fig. 1 E 1002 JCB • VOLUME 172 • NUMBER 7 • 2006 (Fig. 3 B). Similar results (i.e., no difference between wild-type and gei-17 RNAi embryos) were obtained when 75 mM HU was used to trigger a stronger checkpoint response (unpublished data). We conclude that although gei-17 activity reduces the checkpoint response to DNA damage, it has no effect on check- point activation by HU. To examine the effect of loss of gei-17 on checkpoint acti- vation in response to developmental signals, we analyzed the second round of cell division in early embryos. As described in the Introduction, there is a checkpoint-dependent delay in divi- sion of the P1 cell relative to the AB cell during normal develop- ment (Fig. 3 C). The delay normally lasts 2 min; however, when chk-1 is depleted by RNAi, it is reduced to 1 min (Brauchle et al., 2003; Fig. 3 D). If gei-17 negatively controlled the check- point response to developmental signals, we would expect the delay to be extended in gei-17 RNAi embryos, but this was not the case, as gei-17 RNAi embryos showed the same delay as wild type (Fig. 3 D). When MMS was included, however, the lag was signifi cantly extended in gei-17 RNAi embryos and only very modestly extended in wild type (Fig. 3 D). We con- clude that gei-17 functions to suppress checkpoint activity spe- cifi cally in response to DNA damage and not in response to HU-induced stalled replication forks or developmental signals. gei-17 promotes replication fork progression through damaged DNA One explanation for the ability of gei-17 to suppress damage- induced checkpoint activation is that it promotes the rapid replication of damaged DNA. In both X. laevis and yeast, the check point response to MMS-induced damage is known to require the stalling of replication forks (Stokes et al., 2002; Tercero et al., 2003); thus, if gei-17 prevents damage-induced fork stalling, then checkpoint activation would not be expected to occur. To directly assess a role for gei-17 in the replication of damaged DNA, a previously described assay system was used to monitor DNA replication in the early embryo (Edgar and McGhee, 1988; Holway et al., 2005). Egg shells from four-cell Figure 3. gei-17 attenuates checkpoint activation in response to DNA embryos were permeabilized and the samples treated with damage but not HU or developmental signals. (A) Bar graph displaying cytochalasin B to block cytokinesis. The embryos were then the amount of time required for P0 S phase in gei-17 RNAi embryos exposed to control or MMS media and gei-17/chk-1 codepleted embryos exposed to cultured for 1 h before fi xation and DNA staining. Despite the MMS media. The analysis was performed as described in Fig. 1. (B) Bar block to cell division, the DNA replication cycle continues un- graph displaying the amount of time required for P0 S phase in N2 or gei-17 abated, and after 1 h this results in embryos that contain multi- RNAi embryos exposed to media containing 25 mM HU. (C) Schematic de- piction of the effect of checkpoint activation by developmental signals on cell ple nuclei in each of the four cells (Fig. 4 A). The appearance of division in the early embryo. During S phase of the second round of cell divi- multiple nuclei is dependent on DNA synthesis because it does sion, a checkpoint is activated preferentially in the P1 cell (Brauchle et al., not occur in the presence of the replication inhibitors aphidico- 2003). The result is that transition from a two- to three-cell embryo is only briefl y delayed (dotted line), whereas transition from the three- to four-cell em- lin or HU (Edgar and McGhee, 1988; Holway et al., 2005). bryo is more robustly delayed (solid line). The P1-specifi c delay therefore re- MMS did not affect the appearance of multinucleated cells in sults in persistence of a three-cell embryo. (D) Cell division in living embryos wild-type embryos (Fig. 4 B, compare panels II and III). In con- was monitored microscopically, and the time in seconds that the three-cell em- bryo persisted was recorded. Persistence was defi ned as the elapsed time trast, the combination of MMS and gei-17 RNAi caused a de- between division of AB relative to P1 and was assessed for wild-type (N2), fect in DNA replication, as these embryos failed to produce gei-17 RNAi, or chk-1 RNAi embryos. + MMS indicates inclusion of 0.005% multinucleated cells (Fig. 4 B, panel VI). This was not observed MMS. The dotted line represents the endogenous delay that results in part through activation of the checkpoint by developmental signals. n = 15. in undamaged gei-17 RNAi embryos (Fig. 4 B, panel V), dem- onstrating that gei-17 is required for the replication of damaged, but not undamaged, chromosomes. We also note that the repli- and Fig. 3 B). If gei-17 functions to suppress HU-induced cation defect in MMS-exposed gei-17 RNAi embryos was checkpoint activity, we would expect this modest delay to be uniform and occurred in all four cells of the embryo. As the extended in gei-17 RNAi embryos, but this did not occur checkpoint is only highly active in one of these cells (the DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1003 Figure 5. gei-17 prevents replication fork stalling on damaged chromosomes. RAD-51 immunostaining of C. elegans embryos (I, II, IV, and V) or gonads (III and VI) in the presence (IV–VI) or absence (I–III) of MMS (0.005%). Panels I, III, IV, and VI are wild-type embryos or animals, whereas panels II and V are gei-17 RNAi embryos. embryos readily formed RAD-51 foci in response to MMS (Fig. 5, panel V). These data indicate that stalled replication forks, as inferred by the presence of MMS-induced RAD-51 foci, form in cells where MMS triggers the checkpoint (wild-type germ lines and gei-17 RNAi embryos) but not in cells where the checkpoint is silenced (wild-type embryos). RAD-51 foci were not observed in MMS-exposed chk-1 RNAi embryos (unpub- lished data), indicating that attenuation of the checkpoint alone is not suffi cient to explain damage-induced foci formation. Figure 4. gei-17 is required for the replication of damaged but not These results provide further evidence that loss of gei-17 causes undamaged DNA. (A) Schematic depiction of the assay used to monitor replication fork stalling in MMS-exposed embryos. DNA synthesis in early embryos. (B) Four-cell wild-type (panels I–III) or gei-17 RNAi (panels IV–VI) embryos were prepared and cultured as described in the text. The samples were then fi xed and stained with Hoechst 33258 Checkpoint silencing requires the C. elegans after 0 (I and IV) or 60 min, in either the absence (II and V) or presence orthologue of DNA polymerase eta but not (III and VI) of MMS. The images displayed are representative of a group of Rad6 or homologous recombination 20 or more embryos that were examined per sample. All organisms contain mechanisms for promoting the replica- tion of damaged DNA in a manner that does not rely on physical P lineage cell; Brauchle et al., 2003) in intact embryos, this result repair of the lesion. These pathways, termed postreplication suggests that the requirement for gei-17 in the replication of dam- repair or lesion bypass, rely on either translesion synthesis or aged DNA is independent of the checkpoint status of the cell. It is homologous recombination to rescue replication forks that stall possible, however, that permeabilization perturbs the asymmetric at sites of damage (for review see Barbour and Xiao, 2003). distribution of the checkpoint within the four-cell embryo. To explore an involvement of lesion bypass pathways in embryonic The double-strand break repair protein RAD-51 is known checkpoint silencing, we determined the effect of inactivation to accumulate in immunologically detectable foci when replica- of known lesion bypass components on P0 cell cycle progres- tion forks are stalled by DNA damage (Haaf et al., 1995; Scully sion after DNA damage. The role of homologous recombination et al., 1997). The results in Figs. 3 and 4 show that gei-17 is re- was assessed by studying embryos derived from adults carry- quired for S phase progression (Fig. 3) and for DNA replication ing homozygous deletion mutations in the essential recombi- (Fig. 4), specifi cally when chromosomes are damaged. To de- nation genes rad-51 and -54. Neither mutant displayed a defect termine whether loss of gei-17 induces RAD-51 foci, we stained in P0 cell cycle progression after MMS exposure, showing that early embryos (<100 cells) with anti–RAD-51 antibodies homologous recombination is not essential for checkpoint (Colaiacovo et al., 2003). In the absence of MMS, we did not silencing (Fig. 6 A). This is consistent with a lack of RAD-51 detect RAD-51 foci in either wild-type or gei-17 RNAi early foci in MMS-exposed wild-type embryos (Fig. 5). embryos or in the mitotic zone of the hermaphrodite gonad (Fig. 5, The other major lesion bypass pathway in eukaryotes is pan els I–III). In MMS-exposed animals, we could detect robust translesion synthesis, where specialized DNA polymerases are re- RAD-51 foci formation within the mitotic zone of the hermaph- cruited to the replication fork to synthesize DNA across damaged rodite gonad (Fig. 5, panel VI) but not in wild-type early em- bases on the template strand (for review see Prakash et al., 2005). bryos (Fig. 5, panel IV). In contrast to wild type, gei-17 RNAi In yeast and human cells, access of translesion polymerases to 1004 JCB • VOLUME 172 • NUMBER 7 • 2006 Figure 6. Translesion synthesis allows check- point bypass during the early embryonic DNA damage response. (A) The timing of the fi rst cell cycle for embryos of the indicated geno- type was determined as in Fig. 1 E. n = 15. (B) MMS sensitivity was performed as described in Materials and methods for embryos of the indicated genotype. n = 200. n = 15. (C) The timing of the fi rst cell cycle for embryos of the indicated genotype was determined as in Fig. 1 E. (D) polh-1 RNAi embryos were fi xed and stained with anti–RAD-51 antibodies after incubation on either regular (panel I) or MMS- containing (panel II; 0.005% MMS) media. sites of damage is thought to occur through RAD6-mediated ubiq- and the Polκ orthologue polk-1 caused MMS sensitivity in em- uitination of proliferating cell nuclear antigen (PCNA), a DNA bryos (Fig. 6 B). Only polh-1 RNAi, however, delayed progres- replication clamp protein (Hoege et al., 2002; Kannouche et al., sion through P0 S phase (Fig. 6 C). This delay was dependent 2004; Watanabe et al., 2004). Yeast rad6 mutants are sensitive to on MMS and was reversed upon codepletion of chk-1 (Fig. 6 C), MMS and UV light and do not show DNA damage–induced mu- demonstrating that like gei-17 and rad-2, loss of polh-1 allows tagenesis, a hallmark of translesion synthesis (for review see checkpoint activation in the early embryo. Consistent with this, Barbour and Xiao, 2003). To determine whether the Rad6 path- we observed that RAD-51 foci could be detected in polh-1 way is responsible for checkpoint silencing in early embryos, RNAi embryos, in an MMS-dependent manner (Fig. 6 D). we examined the MMS response in ubc-1 mutants. The ubc-1 RAD-51 foci were not observed in MMS-exposed early polk-1 gene represents the sole C. elegans orthologue of budding yeast RNAi embryos (unpublished data). These data indicate that RAD6, and expression of the ubc-1 gene in yeast is suffi cient to polh-1–mediated translesion synthesis is the lesion bypass rescue the rad6 translesion synthesis defect (Leggett et al., 1995). mechanism used by early embryos to silence the checkpoint Surprisingly, embryos derived from adults carrying a homozy- during the DNA damage response. gous deletion of the ubc-1 gene did not display MMS sensitivity (Fig. 6 B) and progressed normally through the P0 cell cycle af- Discussion ter MMS exposure (Fig. 6 C). Thus, in C. elegans embryos, the Rad6 orthologue ubc-1 is not important for the response to Fig. 7 summarizes the findings reported here and integrates MMS-induced damage. them with previous work on cell cycle control in the early It was possible that in C. elegans translesions polymerases embryo. Previous studies have shown that developmental sig- can access sites of damage in a Rad6/ubc-1–independent man- nals, the nature of which are unknown, trigger checkpoint acti- ner. To pursue this hypothesis, we screened all fi ve of the iden- vation and that this contributes to the asynchrony in cell division tifi able translesion polymerases present in C. elegans by RNAi that is required for developmental patterning and germ line for- for MMS sensitivity in embryos. The genes that we screened mation (Fig. 7, shaded portion). Thus, developmental signals included putative orthologues of human Polθ (W03A3.2), Polη represent one class of input into the embryonic checkpoint path- (F53A3.2), Polκ (F22B7.6), Polζ (Y37B11A.2), and Rev1 way. Another type of input is stalled replication (Fig. 7, un- (ZK675.2). The assignment of these C. elegans genes to their shaded portion). Stalled replication has been induced in early putative human counterparts is based purely on sequence con- embryos through mutations in div-1 (Encalada et al., 2000) or servation, as information on the biochemical properties of the through the use of HU (Brauchle et al., 2003; this study). encoded proteins is not available. Of the fi ve, we found that Embryonic sensitivity to stalled replication has been docu- RNAi-mediated depletion of both the Polη orthologue polh-1 mented; it causes checkpoint activation and extends the natural DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1005 is that the checkpoint signal is not strong enough to neutralize the mitosis-promoting capacity of the cytoplasm until the proper ratio is achieved. Thus, in frog and fl y embryos checkpoint avoidance occurs passively. This is in contrast to the active mechanism that we have discovered in C. elegans, and the dif- ference is likely due to when the checkpoint functions during development. In frogs and fl ies the checkpoint is not needed until the midblastula transition, whereas in worms it is used from the fi rst division onward. Rapid embryonic cell cycles occur in all major animal phyla (for review see O’Farrell et al., 2004). C. elegans is no exception, as the early cycles last only 10–40 min. It is possible that the rapid cycling allows no time for lesion repair, and there- fore lesion bypass may be the only viable option for C. elegans embryos exposed to DNA-damaging agents. This is in contrast to the mitotic cells of the C. elegans gonad that can survive delays in cell division and go on to divide normally. Indeed, we have shown that mitotic gonad cells arrest in a checkpoint- dependent manner upon MMS or UV exposure and RAD-51 foci are clearly evident (Figs. 1 and 5 and Table I). Additionally, we were unable to detect any cell cycle arrest phenotype in the germ lines of gei-17 RNAi animals after either high (Table I) or low (not depicted) MMS or UV exposure, suggesting that this pathway is not a major component of the germ line DNA dam- age response. Our results therefore demonstrate a distinct dif- ference between embryonic and germ line responses to DNA damage that could be explained by embryonic sensitivity to the Figure 7. Input/outcome diagram for early embryonic checkpoint timing of cell division. The molecular basis for this difference is responses. Two types of inputs into the embryonic checkpoint are consid- not yet known but likely involves differential expression and/or ered: stalled replication and developmental signals. The shaded portion regulation of members of the gei-17–polh-1 pathway. represents developmentally programmed events, whereas the unshaded portion summarizes how stalled replication can occur and how the embryo Both gei-17 and polh-1 are components of an embryonic prevents it. Arrows represent positive regulation, and the line with a bar on checkpoint silencing pathway that bypasses MMS-induced it represents suppression. Please see Discussion for more details. lesions. The identifi cation of polh-1 as the primary polymerase required for progression through S phase in MMS-exposed asynchrony of cell division (Encalada et al., 2000; Brauchle early embryos is somewhat surprising, as Polη in yeast and et al., 2003). This in turn perturbs development and kills the human cells is primarily associated with UV light–induced em bryo. The focus of the work presented here was on another damage (for review see Prakash et al., 2005). However, budding inducer of stalled replication, DNA damage. We have found that yeast Polη (RAD30) effi ciently bypasses abasic sites (a major early embryos do not stall replication when their chromosomes MMS-induced lesion) when coupled to PCNA in vitro ( Haracska are damaged and that protection against damaged-induced et al., 2001) and is required for maximal abasic site bypass stalled replication is conferred by rad-2, gei-17, and polh-1. in vivo (Zhao et al., 2004). rad30 mutants are accordingly MMS These results explain how the checkpoint can be accessed by sensitive (Zhao et al., 2004). These fi ndings therefore suggest developmental signal–based inputs and insulated from DNA that a role for polh-1 in responding to MMS-induced damage in damage–based inputs. The checkpoint is not insulated from div-1 C. elegans could be explained by the ability of the enzyme to mutant or HU-based inputs, but these are conditions that are bypass abasic sites. irrelevant to wild-type worms in their natural environments. Although the role of polh-1 as a translesion polymerase is Embryonic checkpoint control has been studied in other directly related to replicating damaged DNA, it is not clear what organisms, most notably D. melanogaster and X. laevis. In both role the E3 SUMO ligase, gei-17, actually plays in this process. of these organisms, checkpoints that respond to DNA damage Recent work has shown that SIZ1, which sumoylates PCNA, are not evident until after the rapid cleavage cycles have ended, functions to ward off homologous recombination during lesion at the midblastula transition (Dasso and Newport, 1990; Sibon bypass through recruitment of the RAD51 antagonist SRS2 to the et al., 1997). In X. laevis, the lack of DNA damage checkpoint replication fork (Papouli et al., 2005; Pfander et al., 2005). It is activation in early embryos is likely due to a low DNA/cyto- therefore possible that gei-17 promotes translesion synthesis in plasm ratio, as it has been recently demonstrated that increasing embryos through negative regulation of recombination, although the amount of damaged DNA in younger frog embryos results we do not favor this model, as loss of gei-17 still negatively in a checkpoint-dependent delay in cell division before the mid- affects S phase progression in rad-51 mutant embryos (Fig. 6 A). blastula transition (Conn et al., 2004). The interpretation of this Thus, the elimination of recombination in rad-51 mutants 1006 JCB • VOLUME 172 • NUMBER 7 • 2006 does not suppress the gei-17 RNAi phenotype, and this argues formed on F-1 embryos. For gei-17/chk-1 codepletions, worms were fi rst grown on gei-17 RNAi bacteria for one generation and then moved as against a role for gei-17 in preventing recombination. One pos- F1 L1s onto a plate containing a 1:1 mixture of the feeding vectors. polh-1 sibility is that gei-17 functions in polymerase switching at sites and polk-1 RNAi was accomplished by soaking (Maeda et al., 2001). of DNA damage, and biochemical analysis of the polymerase polh-1/chk-1 codepletions were accomplished by fi rst feeding worms chk-1 bacteria and then soaking P0 L4s in polh-1 double-stranded RNA. Worms switch reaction in C. elegans embryos will be required to deter- were then plated onto regular media or media containing 0.05 mg/ml mine whether this is so. We note that in yeast and mammalian MMS (Sigma-Aldrich), both seeded with chk-1 RNAi bacteria, and analysis cells polymerase switching is controlled by the Rad6 E2 ubiquitin- was performed on their progeny. conjugating enzyme and the Rad18 E3 ubiquitin ligase; however, Analysis of the timing of cell division within living embryos we have shown here that the Rad6 orthologue ubc-1 is not Worms were collected and placed in a drop of M9 buffer for dissection. required for translesion synthesis in C. elegans embryos (Fig. 6, Released embryos were then transferred to agarose pads (2% SeaKem Gold agarose in water) in a small volume of M9 and visualized under B and C), and there is no recognizable Rad18 homologue present Nomarski optics on a microscope (BX51 TF; Olympus). Embryos exposed to in the worm genome. MMS were timed after 20 h of exposure to plates containing 0.05 mg/ml Our results also shed light on the relationship between MMS. Embryos exposed to HU (Calbiochem) were timed after 5 h of expo- sure to plates containing 75 mM HU. Embryos exposed to UV light were checkpoint activation and translesion synthesis, as they suggest timed 1 h after irradiation. Irradiation was performed by placing an open that in the early embryo translesion synthesis trumps check- dish of worms in a Stratalinker (Stratagene). To measure the P0 S phase, point activation to ensure that DNA damage does not slow the timing started when the female pronucleus passed the midline of the embryo. Timing continued until nuclear envelope breakdown had occurred, cell cycle down. How decisions are made at stalled replication just before fi rst mitosis. Because it is unclear when replication initiates, forks to activate one pathway over another is not understood this represents the timing of a partial S phase (Brauchle et al., 2003). and is an active area of research. Our data show that, in the The persistence of three-cell embryos was determined by timing the interval between cytokinesis of the AB cell and cytokinesis of the P1 cell. early embryo, translesion synthesis is so effi cient that check- point activation fails to occur, even when relatively high levels of DNA and antibody staining of embryos and germ lines damage are present. This may reveal a general principle, in that Worms were dissected on glass microscope slides and permeabilized by freeze cracking. Slides were fi xed for 10 min in methanol/formaldehyde during the DNA damage response the default response is to ac- fi xative at −20°C and washed in PBS Tween 20. Slides were then incu- cess the translesion synthesis pathway and that checkpoint acti- bated with anti–RAD-51 antibody (Colaiacovo et al., 2003) at 1:200 over- vation can only occur at levels of damage that saturate translesion night followed by a 2-h incubation in FITC-tagged anti-rabbit secondary antibody. DNA staining was accomplished by adding 10 μl of 10 μg/μl synthesis. Alternatively, embryo-specifi c factors may exist that Hoechst 33258. To count nuclei in the mitotic zone of the gonad, adult allow translesion synthesis to supersede checkpoint activation. worms were fi xed and stained with Hoechst 33258. The distal tip of the The use of the POLH-1 translesion polymerase to prevent gonad was then visualized using fl uorescence microscopy, and the number of nuclei within a constant volume was counted. fork stalling during the early embryonic cell cycles answers the question of how C. elegans embryos bypass checkpoint activation Embryo culture assays and so survive exposure to DNA-damaging agents. Although Embryos were prepared for culturing as described previously (Holway this pathway allows embryonic cells to divide on schedule, et al., 2005). MMS exposure was accomplished by culturing worms for 20 h on 0.05 mg/ml MMS plates and then exposing permeabilized embryos to translesion polymerases are notoriously error prone, and use of 0.2 mg/ml MMS in egg growth media. After incubation, embryos were this pathway predicts that embryos likely trade survival for an stained with Hoechst 33258 and visualized on a microscope. Pictures were increase in mutation frequency. This is especially true when aba- captured using a monochrome camera (SPOT RT; Diagnostic Instruments). sic sites, which are noncoding forms of damage, are considered. MMS sensitivity assays Thus, it appears that during evolution there has been stronger L4 F1 worms grown on plates containing the appropriate bacterial expres- selection for adherence to the schedule of cell division than for sion vectors were transferred to plates containing 0.05 mg/ml MMS. Eggs laid by these worms were collected over time and scored for survival as error-free replication during early embryogenesis, and under- described previously (Holway et al., 2005). standing the basis for this preference will be the goal of future studies in this system. Image acquisition The images shown in Fig. 1 (A–C), Fig. 4 B, Fig. 5, and Fig. 6 D were obtained as follows. All images were collected on a microscope. The type, magnifi cation, and NA of the objective lenses were UPlanAPO, 60× oil, Materials and methods and NA 1.40, respectively. The experiments were performed at room temperature using Hoechst 33258 and FITC-labeled secondary antibodies C. elegans strains and culturing as fl uorochromes. Images were captured on a camera (model 2.1.1; The N2 Bristol strain was used as wild type in all control experiments and Diagnostic Instruments) and processed using SPOT Advanced version for all RNAi experiments. SP482 (rad-3[mn15]), SP488 (rad-2[mn156]), 3.2.4 software (Diagnostic Instruments). TG5 (rad-51[lg8701]), VC531 (rad-54[ok615]), and VC18 (ubc-1[gk14]) strains were obtained from the Caenorhabditis Genetics Center. Animals Online supplemental material were maintained as described previously (Brenner, 1974). Fig. S1 shows that F10G8.7 and gei-17 embryos are sensitive to UV light. The supplemental text describes UV sensitivity assays and the observation that RNAi gei-17 RNAi causes in UV sensitivity in embr yos. Online supplemental material is RNAi by feeding was performed for F10G8.7, W03A3.2, Y37B11A.2, available at http://www.jcb.org/cgi/content/full/jcb.200512136/DC1. ZK675.2, atl-1, gei-17, and chk-1 as described previously (Timmons and Fire, 1998). All bacteria were cultured for 24 h at 37°C in Terrifi c Broth We dedicate this work to John Newport, a pioneer in the study of early containing 50 μg/ml ampicillin, seeded onto nematode growth media embryonic cell cycles. (Brenner, 1974) plates containing 5 mM IPTG, and allowed to dry over- We thank Craig Hunter and Tim Schedl for advice. night. With the exception of chk-1 RNAi, worms were grown for two gen- Some of the strains used in this work were provided by the Caenorhabditis erations on RNAi bacteria. F1 progeny of chk-1 RNAi worms are sterile; Genetics Center, which is funded by the National Institutes of Health therefore, chk-1 RNAi was fed for one generation and analysis was per- National Center for Research Resources. A. La Volpe was supported by a DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1007 MacQueen, A.J., and A.M. Villeneuve. 2001. Nuclear reorganization and homol- Telethon-Italy grant (GGP04010). A.H. Holway was supported by a National ogous chromosome pairing during meiotic prophase require C. elegans Institute of General Medical Sciences (NIGMS) Genetics and Genomics training chk-2. 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Lupardus, P.J., T. Byun, M.C. Yee, M. Hekmat-Nejad, and K.A. Cimprich. 2002. A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev. 16:2327–2332. 1008 JCB • VOLUME 172 • NUMBER 7 • 2006 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png The Journal of Cell Biology Pubmed Central

Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos

Holway, Antonia H.; Kim, Seung-Hwan; La Volpe, Adriana; Michael, W. Matthew
The Journal of Cell Biology , Volume 172 (7) – Mar 27, 2006
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Copyright © 2006, The Rockefeller University Press
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0021-9525
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10.1083/jcb.200512136
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Abstract

JCB: ARTICLE Checkpoint silencing during the DNA damage response in Caenorhabditis elegans embryos 1 1 2 1 Antonia H. Holway, Seung-Hwan Kim, Adriana La Volpe, and W. Matthew Michael The Biological Laboratories, Department of Molecular and Cellular Biology, Harvard University, Cambridge, MA 02138 Istituto di Genetica e Biofi sica “Adriano Buzzati-Traverso,” Consiglio Nazionale delle Ricerche, 80125 Naples, Italy n most cells, the DNA damage checkpoint delays but not in the germ line. Silencing requires rad-2, gei-17, cell division when replication is stalled by DNA and the polh-1 translesion DNA polymerase, which I damage. In early Caenorhabditis elegans embryos, suppress replication fork stalling and thereby eliminate however, the checkpoint responds to developmental the checkpoint-activating signal. These results explain signals that control the timing of cell division, and how checkpoint activation is restricted to developmen- checkpoint activation by nondevelopmental inputs dis- tal signals during embryogenesis and insulated from rupts cell cycle timing and causes embryonic lethality. DNA damage. They also show that checkpoint activa- Given this sensitivity to inappropriate checkpoint acti- tion is not an obligatory response to DNA damage and vation, we were interested in how embryos respond to that pathways exist to bypass the checkpoint when DNA damage. We demonstrate that the checkpoint re- survival depends on uninterrupted progression through sponse to DNA damage is actively silenced in embryos the cell cycle. Introduction Early embryogenesis in many organisms, including Xenopus In embryonic cells, the ATR checkpoint is activated by laevis, Drosophila melanogaster, and Caenorhabditis elegans, endogenous, developmentally programmed cues. The nature of is characterized by rapid progression through the cell cycle (for these signals is not defi ned, but it is clear that developmental review see O’Farrell et al., 2004). Features of early embryonic activation of the checkpoint is important for regulating the tim- cell cycles that distinguish them from somatic cycles include ing of cell division during early embryogenesis. Two examples cell division in the absence of cell growth and a lack of Gap highlight this importance. In D. melanogaster, the mei-41 (ATR) phases. Another important difference between somatic and and grapes (Chk1) genes affect a developmentally programmed embryonic cell cycles concerns the utilization of S phase check- slowing of the cell cycle that occurs at the midblastula transition point pathways. In somatic cells, the S phase checkpoint senses (Sibon et al., 1997, 1999; Su et al., 1999; Yu et al., 2000). Fly DNA damage and responds by delaying progression into mito- embryos perform 13 rounds of rapid and synchronous cell divi- sis (for reviews see Bartek et al., 2004; Sancar et al., 2004). sion before the midblastula transition. After cycle 13, the mei41/ The protein kinases ATR and Chk1 are central to S phase check- grapes checkpoint is activated by an endogenous signal, and point signaling. DNA damage causes replication fork stalling, this slows the cell cycle down. Slowing of the cell cycle in turn which in turn activates ATR and promotes the ATR-directed allows for zygotic transcription to begin, and the control of cell phosphorylation of Chk1. Activated Chk1 delays cell cycle pro- division is thereby transferred from maternal to zygotic regulators. gression through attenuation of core cell cycle regulators such as In mei-41 or grapes mutants, the cell cycle does not slow the Cdc25 protein phosphatase. Thus, in somatic cells, a major down, zygotic control of the cell cycle does not happen on function of the ATR checkpoint is to delay cell cycle progression schedule, and the embryo dies. Therefore, in D. melanogaster, in response to DNA damage until replication can fi nish. the checkpoint plays an important role in remodeling the cell cycle so that zygotic transcription can begin on schedule. Another example of DNA damage–independent utiliza- tion of the ATR checkpoint is found in C. elegans. The one-cell Correspondence to W Correspondence to W.. Matthew Michael: [email protected] Matthew Michael: [email protected] vard.edu embryo, or P0 cell, divides asymmetrically to produce the Abbreviations used in this paper: HU, hydroxyurea; MMS, methanesulphonate; Abbreviations used in this paper: HU, hydroxyurea; MMS, methanesulphonate; smaller (P1) and the larger (AB) daughter cells. The next round PCNA, proliferating cell nuclear antigen; RNAi, RNA inter PCNA, proliferating cell nuclear antigen; RNAi, RNA interference. ference. The online version of this ar The online version of this article contains supplemental material. ticle contains supplemental material. of cell division is asynchronous: AB divides fi rst, followed by © The Rockefeller University Press $8.00 The Journal of Cell Biology, Vol. 172, No. 7, March 27, 2006 999–1008 http://www.jcb.org/cgi/doi/10.1083/jcb.200512136 JCB 999 THE JOURNAL OF CELL BIOLOGY P1 about 2 min later. This 2-min delay is controlled in part through differential activation of the S phase checkpoint in the P1 cell (Brauchle et al., 2003). Developmental checkpoint acti- vation in the early embryo requires the C. elegans homologues of ATR (atl-1) and Chk1 (chk-1). Checkpoint-mediated asyn- chrony in cell division is extremely important to embry- onic patterning in C. elegans. When asynchrony is reduced, through loss of chk-1, the germ line fails to develop and the ani- mal is sterilized (Brauchle et al., 2003; Kalogeropoulos et al., 2004). Extending the asynchrony also has deleterious conse- quences. Hypomorphic mutations in div-1, a gene encoding DNA polymerase α, cause replication problems that result in inappropriate activation of the chk-1 pathway (Encalada et al., 2000; Brauchle et al., 2003). The div-1–mediated activation of chk-1 extends the asynchrony in cell division, and this results in mislocalization of developmental regulators, embryonic patterning defects, and lethality (Encalada et al., 2000). From these examples it is clear that, although checkpoint activation is important for development, it must only occur in response to developmental signals and not in response to un- scheduled events such as replication problems. A common source of replication problems in wild-type cells is DNA damage, and thus it would seem that early embryogenesis in C. elegans would be particularly sensitive to DNA damage be- cause of the deleterious consequences of unscheduled check- point activation. Paradoxically, this is not so, as previous work has shown that wild-type embryos are resistant to relatively high amounts of both UV light and the alkylating agent methyl methanesulphonate (MMS; Hartman and Herman, 1982; Holway et al., 2005), two DNA-damaging agents that are known to cause replication problems and subsequent check- point activation (Lupardus et al., 2002; Stokes et al., 2002; Tercero et al., 2003). We resolve this paradox by showing that the checkpoint is actively silenced during the DNA damage response in early embryos. We go on to defi ne genetic require- ments and the basis for checkpoint silencing. Our results identify a novel developmental mechanism that ensures that Figure 1. Differential checkpoint responses to DNA damage in the germ line cell cycle progression is not attenuated by DNA damage, thus and early embryo. (A–C) Gonads were dissected from adult hermaphrodites, providing embryos with a chance of survival even when their fi xed, and stained with Hoechst 33258 to visualize the nuclei. Where indicated, the worms had been exposed to MMS (B and C; 0.005% MMS) chromosomes are heavily damaged. or atl-1 RNAi (C) before fi xation. Nuclei were then visualized and photographed by fl uorescence microscopy. (D) Schematic depiction of the fi rst cell cycle during C. elegans embryogenesis. (E) Wild-type (N2) em- Results bryos were cultured on regular media (control), media containing 75 mM HU (HU), media containing HU and E. coli expressing double-stranded Levels of DNA-damaging agents that RNA against chk-1 (+ HU + chk-1 RNAi), or media containing 0.005% trigger a checkpoint arrest in germ cells MMS (+ MMS) or were exposed to 100 J/m of UV light (+ UV). The tim- ing of the fi rst embryonic cell cycle was then determined by microscopic do not activate the checkpoint in embryos examination of living embryos, and the mean time, from a minimum of It was not known whether the C. elegans checkpoint pathway 10 samples per data point, for P0 S phase progression is displayed. can sense the types of DNA damage that cause replication stress, P0 S phase progression is defi ned as the elapsed time required to progress from step ii to v in the diagram in D. Error bars represent one standard such as alkylation or UV light–induced damage. Previous work deviation from the mean. (F) Same as E, except that the media contained has shown that nuclei in the mitotic zone of the hermaphrodite the indicated concentrations of MMS. gonad, a nonembryonic tissue, undergo checkpoint-dependent cell cycle arrest in response to replication blocks and ionizing radiation (Gartner et al., 2000; MacQueen and Villeneuve, Hoechst 33258 to visualize nuclei. MMS reduced the number 2001). This arrest is refl ected by a reduction in nuclei number of nuclei within the mitotic zone from a mean of 35 to a mean and an increase in nuclear size. To see whether MMS and/or UV of 22 (Fig. 1, A and B; and Table I). The effect of MMS in light induced checkpoint activation in the gonad, animals were the germ line was reversed when the checkpoint gene atl-1, exposed to 0.005% MMS and then fi xed and stained with the worm orthologue of ATR (Brauchle et al., 2003), was 1000 JCB • VOLUME 172 • NUMBER 7 • 2006 Table I. Nuclei in the mitotic zone of the hermaphrodite gonad Table II. Embryonic lethality after chronic MMS exposure Genotype Condition Nuclei count MMS Time Emb %h % Wild type Control 35 ± 2.6 —16 0 Wild type MMS 22.5 ± 2.0 —32 0 Wild type UV 20.9 ± 3.5 0.001 16 0 atl-1 RNAi Control 34.1 ± 2.9 0.001 32 0 atl-1 RNAi MMS 33.4 ± 4.0 0.005 16 1.3 atl-1 RNAi UV 34.8 ± 4.3 0.005 32 2.4 gei-17 RNAi Control 34.6 ± 3.9 0.01 16 73 gei-17 RNAi MMS 22.6 ± 3.1 0.01 32 94 gei-17 RNAi UV 0.025 16 96 21.0 ± 2.4 0.025 32 98 Young adult hermaphrodites of the indicated genotype were fi xed and stained with Hoechst 33258 to visualize nuclei in the mitotic zone of the gonad. The Young adult hermaphrodites were transferred to media containing the indicated nuclei within a fi xed volume were then counted for a minimum of 10 samples per concentration of MMS. After 16 h, the animals were transferred to fresh MMS data point. Shown are these counts with the standard deviation. Condition refers plates, and incubation was continued for an additional 16 h. At the end of to animals that were not exposed to DNA-damaging agents (control), animals each 16-h incubation, the eggs that had been laid were counted and then that were exposed to 0.005% MMS (MMS), or animals that were exposed to counted again 20 h later. Emb refers to the percentage of embryonic lethality 100 J/m of UV light (UV). or the percentage of eggs that failed to hatch during the 20 h after removal of the adults. depleted by RNA interference (RNAi; Fig. 1 C and Table I). Similar results were obtained when animals were irradiated of embryos to avoid a checkpoint response to MMS is saturable. with 100 J/m of UV light (Table I). We conclude that germ They also indicate that checkpoint silencing and survival of DNA cells undergo checkpoint-dependent cell cycle arrest upon damage are linked, and this is consistent with previous work exposure to either MMS or UV light and that, therefore, the showing that even modest perturbations in the timing of cell divi- C. elegans checkpoint can indeed sense MMS- and UV light– sion are lethal to the developing embryo (Encalada et al., 2000). induced damage. The effect of MMS and UV light on cell cycle progression Embryonic checkpoint silencing in the early embryo was examined next. Fig. 1 D shows the is independent of lesion repair major events of the fi rst mitotic interphase in the early embryo. yet under genetic control After fertilization (step i), the female pronucleus migrates across A simple explanation for checkpoint silencing is that embryos the embryo, or P0 cell, where it meets and apposes the male rapidly repair damaged DNA. However, extensive analysis of pronucleus (steps ii–iv). DNA replication then fi nishes, and mi- the kinetics of DNA repair in C. elegans has been reported tosis is initiated by nuclear envelope breakdown (step v). Previ- (Hartman and Herman, 1982; Hartman, 1984; Hartman et al., ous work has shown that replication stress–induced checkpoint 1989; Jones and Hartman, 1996), and these studies demonstrate activation, as triggered by the replication inhibitor hydroxyurea that repair is unlikely to account for checkpoint silencing in the (HU), occurs at the one-cell stage (Brauchle et al., 2003). This embryo. For example, >80% of (6-4) photoproducts remain in checkpoint requires the chk-1 gene (Brauchle et al., 2003) and embryos 3 h after a dose of 50 J/m of UV light is delivered prevents the transition from step iv to v in Fig. 1 D. Animals (Hartman et al., 1989). The data in Fig. 1 E were collected 1 h were exposed to MMS, UV light, or, as a positive control, HU, after a dose of 100 J/m was delivered, and thus the embryo and the timing of the fi rst cell cycle was determined by direct could not possibly have repaired even a modest percentage of microscopic visualization of living embryos. As shown in Fig. 1 E, the damage in that short a period of time. We conclude that cell and as previously reported (Brauchle et al., 2003), when em- cycle progression occurs unimpeded even when the level of bryos were treated with HU, there was a signifi cant delay in damage present greatly exceeds the capacity of the embryo to progression through the P0 cell cycle. This delay was check- repair it. point dependent, as it was reversed after depletion of chk-1 by If embryonic cell cycle progression is truly independent RNAi. In contrast to HU, we did not detect a signifi cant P0 cell of repair, then mutant embryos that are defi cient in DNA repair cycle delay when the embryos were exposed to MMS or UV would nonetheless exhibit normal cell cycles after DNA damage. light (Fig. 1 E). We conclude that the amounts of MMS or UV To test this, we examined cell cycle progression in early rad-3 light that are suf cient to acti fi vate a checkpoint response in the embryos. rad-3 mutant embryos have a defect in excision repair germ line (0.005% and 100 J/m , respectively) cannot do so in and are consequently very sensitive to both MMS (Fig. 2 A) and early embryos. We refer to this phenomenon as early embryonic UV light (Hartman and Herman, 1982; Hartman et al., 1989). checkpoint silencing. The rate of repair in rad-3 embryos has been determined and is We next tested how much MMS embryos could endure threefold lower than wild type (Hartman et al., 1989). Despite before a delay in cell division was detected. For this, we timed this reduced capacity for repair, however, the timing of cell the fi rst cell division after exposure to a range of MMS concen- division in rad-3 mutant embryos was indistinguishable from trations and found that concentrations >0.005% caused both a wild type after exposure to either UV light or MMS (Fig. 2 B). delay in progression through S phase (Fig. 1 F) and high levels The dose of UV light used in the experiment is suffi cient to of embryonic lethality (Table II). These data show that the ability kill 100% of the rad-3 mutant embryos and <10% of wild-type DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1001 indistinguishable from wild type (Hartman, 1984). Thus repair- defi cient rad-3 mutants have normal cell cycles after DNA damage, whereas repair-profi cient rad-2 mutants do not. This shows that a process that is independent of DNA repair is responsible for preventing checkpoint activation during the early embryonic cell cycle. Consistent with this, we also found that RNAi-mediated depletion of another excision repair gene, the C. elegans homologue of the human XPF endonuclease (F10G8.7), renders embryos extremely sensitive to both UV light (Fig. S1, avail- able at http://www.jcb.org/cgi/content/full/jcb.200512136) and MMS (Fig. 2 A) yet had no affect on cell cycle progression in the early embryo (Fig. 2 B). We conclude that checkpoint silencing is independent of lesion repair (based on the results with rad-3 and F10G8.7) yet nonetheless under genetic control (based on the results with rad-2). gei-17 suppresses the checkpoint response to DNA damage but not developmental signals or stalled replication forks in early embryos The fi nding that checkpoint silencing is under genetic control prompted a search for genes that silence the checkpoint when DNA damage is present. The rad-2 gene has not yet been cloned, and we are currently working toward accomplishing this. Recent work from our laboratory has shown that the gei-17 gene, which encodes an E3 SUMO ligase related to yeast SIZ1 and human PIAS1, is an important participant in the embryonic DNA damage response in C. elegans (Holway et al., 2005). Depletion of gei-17 by RNAi renders embryos sensitive to both MMS (Holway et al., 2005) and UV light (Fig. S1). MMS- exposed gei-17 RNAi early embryos display abnormal nuclear morphology, characterized by fused nuclei and anaphase bridg- ing (Holway et al., 2005). These results suggested that gei-17 is important for early embryonic cell cycle progression when dam- age is present and prompted us to examine the kinetics of cell division in gei-17 RNAi early embryos. MMS exposure delayed progression through the P0 S phase in gei-17 RNAi embryos. At 0.005% MMS, we observed an 440-s delay (Fig. 3 A) and at 0.001% MMS the delay was 300 s (not depicted). These data Figure 2. Normal progression through the early cell cycles in excision demonstrate that S phase takes longer in gei-17 RNAi embryos repair–defi cient embryos exposed to DNA damage. (A) MMS sensitivity exposed to 0.001% MMS than it does in wild-type embryos of embryos of the indicated genotype. Details on the MMS sensitivity assay can be found in Materials and methods and in Holway et al. (2005). exposed to 10-fold more MMS (the wild-type delay at 0.01% (B) Timing of the P0 S phase was determined as in Fig. 1 E for embryos of MMS was 120 s; Fig. 1 F). To determine whether these MMS- the indicated genotype. Control refers to regular media, + MMS refers to induced effects were caused by activation of the checkpoint, we media containing 0.005% MMS, and + UV refers to exposure to 100 J/m of UV light. codepleted gei-17 with chk-1. As was the case with rad-2, code- pletion of gei-17 with chk-1 reversed the MMS-induced delay in progression through the P0 cell cycle (Fig. 3 A). This result embryos (unpublished data; see Hartman and Herman [1982] demonstrates that gei-17 activity suppresses checkpoint activa- for UV sensitivity of rad-3 mutants). Interestingly, another tion in response to DNA damage in the early embryo. radiation- and MMS-sensitive mutant, rad-2, showed altered To see whether the effect of gei-17 on checkpoint activa- progression through the fi rst cell cycle after exposure to UV or tion was specifi c for DNA damage, we next examined check- MMS (Fig. 2, A and B). rad-2 mutant embryos delayed progres- point activation in gei-17 RNAi embryos in response to both sion through the P0 S phase in a manner that was dependent on HU and developmental signals. For the HU experiment, we DNA damage and similar to wild-type embryos exposed to HU. used a lower concentration of HU than that used in Fig. 1 The damage-induced delay in rad-2 embryos was checkpoint (25 as opposed to 75 mM), and this resulted in a more modest dependent, as it was reversed when chk-1 was depleted by RNAi delay in cell division in wild-type embryos (160 s delay after (Fig. 2 B). Importantly, the rate of repair in rad-2 mutants is 25 mM HU in contrast to the 475-s delay after 75 mM; Fig. 1 E 1002 JCB • VOLUME 172 • NUMBER 7 • 2006 (Fig. 3 B). Similar results (i.e., no difference between wild-type and gei-17 RNAi embryos) were obtained when 75 mM HU was used to trigger a stronger checkpoint response (unpublished data). We conclude that although gei-17 activity reduces the checkpoint response to DNA damage, it has no effect on check- point activation by HU. To examine the effect of loss of gei-17 on checkpoint acti- vation in response to developmental signals, we analyzed the second round of cell division in early embryos. As described in the Introduction, there is a checkpoint-dependent delay in divi- sion of the P1 cell relative to the AB cell during normal develop- ment (Fig. 3 C). The delay normally lasts 2 min; however, when chk-1 is depleted by RNAi, it is reduced to 1 min (Brauchle et al., 2003; Fig. 3 D). If gei-17 negatively controlled the check- point response to developmental signals, we would expect the delay to be extended in gei-17 RNAi embryos, but this was not the case, as gei-17 RNAi embryos showed the same delay as wild type (Fig. 3 D). When MMS was included, however, the lag was signifi cantly extended in gei-17 RNAi embryos and only very modestly extended in wild type (Fig. 3 D). We con- clude that gei-17 functions to suppress checkpoint activity spe- cifi cally in response to DNA damage and not in response to HU-induced stalled replication forks or developmental signals. gei-17 promotes replication fork progression through damaged DNA One explanation for the ability of gei-17 to suppress damage- induced checkpoint activation is that it promotes the rapid replication of damaged DNA. In both X. laevis and yeast, the check point response to MMS-induced damage is known to require the stalling of replication forks (Stokes et al., 2002; Tercero et al., 2003); thus, if gei-17 prevents damage-induced fork stalling, then checkpoint activation would not be expected to occur. To directly assess a role for gei-17 in the replication of damaged DNA, a previously described assay system was used to monitor DNA replication in the early embryo (Edgar and McGhee, 1988; Holway et al., 2005). Egg shells from four-cell Figure 3. gei-17 attenuates checkpoint activation in response to DNA embryos were permeabilized and the samples treated with damage but not HU or developmental signals. (A) Bar graph displaying cytochalasin B to block cytokinesis. The embryos were then the amount of time required for P0 S phase in gei-17 RNAi embryos exposed to control or MMS media and gei-17/chk-1 codepleted embryos exposed to cultured for 1 h before fi xation and DNA staining. Despite the MMS media. The analysis was performed as described in Fig. 1. (B) Bar block to cell division, the DNA replication cycle continues un- graph displaying the amount of time required for P0 S phase in N2 or gei-17 abated, and after 1 h this results in embryos that contain multi- RNAi embryos exposed to media containing 25 mM HU. (C) Schematic de- piction of the effect of checkpoint activation by developmental signals on cell ple nuclei in each of the four cells (Fig. 4 A). The appearance of division in the early embryo. During S phase of the second round of cell divi- multiple nuclei is dependent on DNA synthesis because it does sion, a checkpoint is activated preferentially in the P1 cell (Brauchle et al., not occur in the presence of the replication inhibitors aphidico- 2003). The result is that transition from a two- to three-cell embryo is only briefl y delayed (dotted line), whereas transition from the three- to four-cell em- lin or HU (Edgar and McGhee, 1988; Holway et al., 2005). bryo is more robustly delayed (solid line). The P1-specifi c delay therefore re- MMS did not affect the appearance of multinucleated cells in sults in persistence of a three-cell embryo. (D) Cell division in living embryos wild-type embryos (Fig. 4 B, compare panels II and III). In con- was monitored microscopically, and the time in seconds that the three-cell em- bryo persisted was recorded. Persistence was defi ned as the elapsed time trast, the combination of MMS and gei-17 RNAi caused a de- between division of AB relative to P1 and was assessed for wild-type (N2), fect in DNA replication, as these embryos failed to produce gei-17 RNAi, or chk-1 RNAi embryos. + MMS indicates inclusion of 0.005% multinucleated cells (Fig. 4 B, panel VI). This was not observed MMS. The dotted line represents the endogenous delay that results in part through activation of the checkpoint by developmental signals. n = 15. in undamaged gei-17 RNAi embryos (Fig. 4 B, panel V), dem- onstrating that gei-17 is required for the replication of damaged, but not undamaged, chromosomes. We also note that the repli- and Fig. 3 B). If gei-17 functions to suppress HU-induced cation defect in MMS-exposed gei-17 RNAi embryos was checkpoint activity, we would expect this modest delay to be uniform and occurred in all four cells of the embryo. As the extended in gei-17 RNAi embryos, but this did not occur checkpoint is only highly active in one of these cells (the DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1003 Figure 5. gei-17 prevents replication fork stalling on damaged chromosomes. RAD-51 immunostaining of C. elegans embryos (I, II, IV, and V) or gonads (III and VI) in the presence (IV–VI) or absence (I–III) of MMS (0.005%). Panels I, III, IV, and VI are wild-type embryos or animals, whereas panels II and V are gei-17 RNAi embryos. embryos readily formed RAD-51 foci in response to MMS (Fig. 5, panel V). These data indicate that stalled replication forks, as inferred by the presence of MMS-induced RAD-51 foci, form in cells where MMS triggers the checkpoint (wild-type germ lines and gei-17 RNAi embryos) but not in cells where the checkpoint is silenced (wild-type embryos). RAD-51 foci were not observed in MMS-exposed chk-1 RNAi embryos (unpub- lished data), indicating that attenuation of the checkpoint alone is not suffi cient to explain damage-induced foci formation. Figure 4. gei-17 is required for the replication of damaged but not These results provide further evidence that loss of gei-17 causes undamaged DNA. (A) Schematic depiction of the assay used to monitor replication fork stalling in MMS-exposed embryos. DNA synthesis in early embryos. (B) Four-cell wild-type (panels I–III) or gei-17 RNAi (panels IV–VI) embryos were prepared and cultured as described in the text. The samples were then fi xed and stained with Hoechst 33258 Checkpoint silencing requires the C. elegans after 0 (I and IV) or 60 min, in either the absence (II and V) or presence orthologue of DNA polymerase eta but not (III and VI) of MMS. The images displayed are representative of a group of Rad6 or homologous recombination 20 or more embryos that were examined per sample. All organisms contain mechanisms for promoting the replica- tion of damaged DNA in a manner that does not rely on physical P lineage cell; Brauchle et al., 2003) in intact embryos, this result repair of the lesion. These pathways, termed postreplication suggests that the requirement for gei-17 in the replication of dam- repair or lesion bypass, rely on either translesion synthesis or aged DNA is independent of the checkpoint status of the cell. It is homologous recombination to rescue replication forks that stall possible, however, that permeabilization perturbs the asymmetric at sites of damage (for review see Barbour and Xiao, 2003). distribution of the checkpoint within the four-cell embryo. To explore an involvement of lesion bypass pathways in embryonic The double-strand break repair protein RAD-51 is known checkpoint silencing, we determined the effect of inactivation to accumulate in immunologically detectable foci when replica- of known lesion bypass components on P0 cell cycle progres- tion forks are stalled by DNA damage (Haaf et al., 1995; Scully sion after DNA damage. The role of homologous recombination et al., 1997). The results in Figs. 3 and 4 show that gei-17 is re- was assessed by studying embryos derived from adults carry- quired for S phase progression (Fig. 3) and for DNA replication ing homozygous deletion mutations in the essential recombi- (Fig. 4), specifi cally when chromosomes are damaged. To de- nation genes rad-51 and -54. Neither mutant displayed a defect termine whether loss of gei-17 induces RAD-51 foci, we stained in P0 cell cycle progression after MMS exposure, showing that early embryos (<100 cells) with anti–RAD-51 antibodies homologous recombination is not essential for checkpoint (Colaiacovo et al., 2003). In the absence of MMS, we did not silencing (Fig. 6 A). This is consistent with a lack of RAD-51 detect RAD-51 foci in either wild-type or gei-17 RNAi early foci in MMS-exposed wild-type embryos (Fig. 5). embryos or in the mitotic zone of the hermaphrodite gonad (Fig. 5, The other major lesion bypass pathway in eukaryotes is pan els I–III). In MMS-exposed animals, we could detect robust translesion synthesis, where specialized DNA polymerases are re- RAD-51 foci formation within the mitotic zone of the hermaph- cruited to the replication fork to synthesize DNA across damaged rodite gonad (Fig. 5, panel VI) but not in wild-type early em- bases on the template strand (for review see Prakash et al., 2005). bryos (Fig. 5, panel IV). In contrast to wild type, gei-17 RNAi In yeast and human cells, access of translesion polymerases to 1004 JCB • VOLUME 172 • NUMBER 7 • 2006 Figure 6. Translesion synthesis allows check- point bypass during the early embryonic DNA damage response. (A) The timing of the fi rst cell cycle for embryos of the indicated geno- type was determined as in Fig. 1 E. n = 15. (B) MMS sensitivity was performed as described in Materials and methods for embryos of the indicated genotype. n = 200. n = 15. (C) The timing of the fi rst cell cycle for embryos of the indicated genotype was determined as in Fig. 1 E. (D) polh-1 RNAi embryos were fi xed and stained with anti–RAD-51 antibodies after incubation on either regular (panel I) or MMS- containing (panel II; 0.005% MMS) media. sites of damage is thought to occur through RAD6-mediated ubiq- and the Polκ orthologue polk-1 caused MMS sensitivity in em- uitination of proliferating cell nuclear antigen (PCNA), a DNA bryos (Fig. 6 B). Only polh-1 RNAi, however, delayed progres- replication clamp protein (Hoege et al., 2002; Kannouche et al., sion through P0 S phase (Fig. 6 C). This delay was dependent 2004; Watanabe et al., 2004). Yeast rad6 mutants are sensitive to on MMS and was reversed upon codepletion of chk-1 (Fig. 6 C), MMS and UV light and do not show DNA damage–induced mu- demonstrating that like gei-17 and rad-2, loss of polh-1 allows tagenesis, a hallmark of translesion synthesis (for review see checkpoint activation in the early embryo. Consistent with this, Barbour and Xiao, 2003). To determine whether the Rad6 path- we observed that RAD-51 foci could be detected in polh-1 way is responsible for checkpoint silencing in early embryos, RNAi embryos, in an MMS-dependent manner (Fig. 6 D). we examined the MMS response in ubc-1 mutants. The ubc-1 RAD-51 foci were not observed in MMS-exposed early polk-1 gene represents the sole C. elegans orthologue of budding yeast RNAi embryos (unpublished data). These data indicate that RAD6, and expression of the ubc-1 gene in yeast is suffi cient to polh-1–mediated translesion synthesis is the lesion bypass rescue the rad6 translesion synthesis defect (Leggett et al., 1995). mechanism used by early embryos to silence the checkpoint Surprisingly, embryos derived from adults carrying a homozy- during the DNA damage response. gous deletion of the ubc-1 gene did not display MMS sensitivity (Fig. 6 B) and progressed normally through the P0 cell cycle af- Discussion ter MMS exposure (Fig. 6 C). Thus, in C. elegans embryos, the Rad6 orthologue ubc-1 is not important for the response to Fig. 7 summarizes the findings reported here and integrates MMS-induced damage. them with previous work on cell cycle control in the early It was possible that in C. elegans translesions polymerases embryo. Previous studies have shown that developmental sig- can access sites of damage in a Rad6/ubc-1–independent man- nals, the nature of which are unknown, trigger checkpoint acti- ner. To pursue this hypothesis, we screened all fi ve of the iden- vation and that this contributes to the asynchrony in cell division tifi able translesion polymerases present in C. elegans by RNAi that is required for developmental patterning and germ line for- for MMS sensitivity in embryos. The genes that we screened mation (Fig. 7, shaded portion). Thus, developmental signals included putative orthologues of human Polθ (W03A3.2), Polη represent one class of input into the embryonic checkpoint path- (F53A3.2), Polκ (F22B7.6), Polζ (Y37B11A.2), and Rev1 way. Another type of input is stalled replication (Fig. 7, un- (ZK675.2). The assignment of these C. elegans genes to their shaded portion). Stalled replication has been induced in early putative human counterparts is based purely on sequence con- embryos through mutations in div-1 (Encalada et al., 2000) or servation, as information on the biochemical properties of the through the use of HU (Brauchle et al., 2003; this study). encoded proteins is not available. Of the fi ve, we found that Embryonic sensitivity to stalled replication has been docu- RNAi-mediated depletion of both the Polη orthologue polh-1 mented; it causes checkpoint activation and extends the natural DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1005 is that the checkpoint signal is not strong enough to neutralize the mitosis-promoting capacity of the cytoplasm until the proper ratio is achieved. Thus, in frog and fl y embryos checkpoint avoidance occurs passively. This is in contrast to the active mechanism that we have discovered in C. elegans, and the dif- ference is likely due to when the checkpoint functions during development. In frogs and fl ies the checkpoint is not needed until the midblastula transition, whereas in worms it is used from the fi rst division onward. Rapid embryonic cell cycles occur in all major animal phyla (for review see O’Farrell et al., 2004). C. elegans is no exception, as the early cycles last only 10–40 min. It is possible that the rapid cycling allows no time for lesion repair, and there- fore lesion bypass may be the only viable option for C. elegans embryos exposed to DNA-damaging agents. This is in contrast to the mitotic cells of the C. elegans gonad that can survive delays in cell division and go on to divide normally. Indeed, we have shown that mitotic gonad cells arrest in a checkpoint- dependent manner upon MMS or UV exposure and RAD-51 foci are clearly evident (Figs. 1 and 5 and Table I). Additionally, we were unable to detect any cell cycle arrest phenotype in the germ lines of gei-17 RNAi animals after either high (Table I) or low (not depicted) MMS or UV exposure, suggesting that this pathway is not a major component of the germ line DNA dam- age response. Our results therefore demonstrate a distinct dif- ference between embryonic and germ line responses to DNA damage that could be explained by embryonic sensitivity to the Figure 7. Input/outcome diagram for early embryonic checkpoint timing of cell division. The molecular basis for this difference is responses. Two types of inputs into the embryonic checkpoint are consid- not yet known but likely involves differential expression and/or ered: stalled replication and developmental signals. The shaded portion regulation of members of the gei-17–polh-1 pathway. represents developmentally programmed events, whereas the unshaded portion summarizes how stalled replication can occur and how the embryo Both gei-17 and polh-1 are components of an embryonic prevents it. Arrows represent positive regulation, and the line with a bar on checkpoint silencing pathway that bypasses MMS-induced it represents suppression. Please see Discussion for more details. lesions. The identifi cation of polh-1 as the primary polymerase required for progression through S phase in MMS-exposed asynchrony of cell division (Encalada et al., 2000; Brauchle early embryos is somewhat surprising, as Polη in yeast and et al., 2003). This in turn perturbs development and kills the human cells is primarily associated with UV light–induced em bryo. The focus of the work presented here was on another damage (for review see Prakash et al., 2005). However, budding inducer of stalled replication, DNA damage. We have found that yeast Polη (RAD30) effi ciently bypasses abasic sites (a major early embryos do not stall replication when their chromosomes MMS-induced lesion) when coupled to PCNA in vitro ( Haracska are damaged and that protection against damaged-induced et al., 2001) and is required for maximal abasic site bypass stalled replication is conferred by rad-2, gei-17, and polh-1. in vivo (Zhao et al., 2004). rad30 mutants are accordingly MMS These results explain how the checkpoint can be accessed by sensitive (Zhao et al., 2004). These fi ndings therefore suggest developmental signal–based inputs and insulated from DNA that a role for polh-1 in responding to MMS-induced damage in damage–based inputs. The checkpoint is not insulated from div-1 C. elegans could be explained by the ability of the enzyme to mutant or HU-based inputs, but these are conditions that are bypass abasic sites. irrelevant to wild-type worms in their natural environments. Although the role of polh-1 as a translesion polymerase is Embryonic checkpoint control has been studied in other directly related to replicating damaged DNA, it is not clear what organisms, most notably D. melanogaster and X. laevis. In both role the E3 SUMO ligase, gei-17, actually plays in this process. of these organisms, checkpoints that respond to DNA damage Recent work has shown that SIZ1, which sumoylates PCNA, are not evident until after the rapid cleavage cycles have ended, functions to ward off homologous recombination during lesion at the midblastula transition (Dasso and Newport, 1990; Sibon bypass through recruitment of the RAD51 antagonist SRS2 to the et al., 1997). In X. laevis, the lack of DNA damage checkpoint replication fork (Papouli et al., 2005; Pfander et al., 2005). It is activation in early embryos is likely due to a low DNA/cyto- therefore possible that gei-17 promotes translesion synthesis in plasm ratio, as it has been recently demonstrated that increasing embryos through negative regulation of recombination, although the amount of damaged DNA in younger frog embryos results we do not favor this model, as loss of gei-17 still negatively in a checkpoint-dependent delay in cell division before the mid- affects S phase progression in rad-51 mutant embryos (Fig. 6 A). blastula transition (Conn et al., 2004). The interpretation of this Thus, the elimination of recombination in rad-51 mutants 1006 JCB • VOLUME 172 • NUMBER 7 • 2006 does not suppress the gei-17 RNAi phenotype, and this argues formed on F-1 embryos. For gei-17/chk-1 codepletions, worms were fi rst grown on gei-17 RNAi bacteria for one generation and then moved as against a role for gei-17 in preventing recombination. One pos- F1 L1s onto a plate containing a 1:1 mixture of the feeding vectors. polh-1 sibility is that gei-17 functions in polymerase switching at sites and polk-1 RNAi was accomplished by soaking (Maeda et al., 2001). of DNA damage, and biochemical analysis of the polymerase polh-1/chk-1 codepletions were accomplished by fi rst feeding worms chk-1 bacteria and then soaking P0 L4s in polh-1 double-stranded RNA. Worms switch reaction in C. elegans embryos will be required to deter- were then plated onto regular media or media containing 0.05 mg/ml mine whether this is so. We note that in yeast and mammalian MMS (Sigma-Aldrich), both seeded with chk-1 RNAi bacteria, and analysis cells polymerase switching is controlled by the Rad6 E2 ubiquitin- was performed on their progeny. conjugating enzyme and the Rad18 E3 ubiquitin ligase; however, Analysis of the timing of cell division within living embryos we have shown here that the Rad6 orthologue ubc-1 is not Worms were collected and placed in a drop of M9 buffer for dissection. required for translesion synthesis in C. elegans embryos (Fig. 6, Released embryos were then transferred to agarose pads (2% SeaKem Gold agarose in water) in a small volume of M9 and visualized under B and C), and there is no recognizable Rad18 homologue present Nomarski optics on a microscope (BX51 TF; Olympus). Embryos exposed to in the worm genome. MMS were timed after 20 h of exposure to plates containing 0.05 mg/ml Our results also shed light on the relationship between MMS. Embryos exposed to HU (Calbiochem) were timed after 5 h of expo- sure to plates containing 75 mM HU. Embryos exposed to UV light were checkpoint activation and translesion synthesis, as they suggest timed 1 h after irradiation. Irradiation was performed by placing an open that in the early embryo translesion synthesis trumps check- dish of worms in a Stratalinker (Stratagene). To measure the P0 S phase, point activation to ensure that DNA damage does not slow the timing started when the female pronucleus passed the midline of the embryo. Timing continued until nuclear envelope breakdown had occurred, cell cycle down. How decisions are made at stalled replication just before fi rst mitosis. Because it is unclear when replication initiates, forks to activate one pathway over another is not understood this represents the timing of a partial S phase (Brauchle et al., 2003). and is an active area of research. Our data show that, in the The persistence of three-cell embryos was determined by timing the interval between cytokinesis of the AB cell and cytokinesis of the P1 cell. early embryo, translesion synthesis is so effi cient that check- point activation fails to occur, even when relatively high levels of DNA and antibody staining of embryos and germ lines damage are present. This may reveal a general principle, in that Worms were dissected on glass microscope slides and permeabilized by freeze cracking. Slides were fi xed for 10 min in methanol/formaldehyde during the DNA damage response the default response is to ac- fi xative at −20°C and washed in PBS Tween 20. Slides were then incu- cess the translesion synthesis pathway and that checkpoint acti- bated with anti–RAD-51 antibody (Colaiacovo et al., 2003) at 1:200 over- vation can only occur at levels of damage that saturate translesion night followed by a 2-h incubation in FITC-tagged anti-rabbit secondary antibody. DNA staining was accomplished by adding 10 μl of 10 μg/μl synthesis. Alternatively, embryo-specifi c factors may exist that Hoechst 33258. To count nuclei in the mitotic zone of the gonad, adult allow translesion synthesis to supersede checkpoint activation. worms were fi xed and stained with Hoechst 33258. The distal tip of the The use of the POLH-1 translesion polymerase to prevent gonad was then visualized using fl uorescence microscopy, and the number of nuclei within a constant volume was counted. fork stalling during the early embryonic cell cycles answers the question of how C. elegans embryos bypass checkpoint activation Embryo culture assays and so survive exposure to DNA-damaging agents. Although Embryos were prepared for culturing as described previously (Holway this pathway allows embryonic cells to divide on schedule, et al., 2005). MMS exposure was accomplished by culturing worms for 20 h on 0.05 mg/ml MMS plates and then exposing permeabilized embryos to translesion polymerases are notoriously error prone, and use of 0.2 mg/ml MMS in egg growth media. After incubation, embryos were this pathway predicts that embryos likely trade survival for an stained with Hoechst 33258 and visualized on a microscope. Pictures were increase in mutation frequency. This is especially true when aba- captured using a monochrome camera (SPOT RT; Diagnostic Instruments). sic sites, which are noncoding forms of damage, are considered. MMS sensitivity assays Thus, it appears that during evolution there has been stronger L4 F1 worms grown on plates containing the appropriate bacterial expres- selection for adherence to the schedule of cell division than for sion vectors were transferred to plates containing 0.05 mg/ml MMS. Eggs laid by these worms were collected over time and scored for survival as error-free replication during early embryogenesis, and under- described previously (Holway et al., 2005). standing the basis for this preference will be the goal of future studies in this system. Image acquisition The images shown in Fig. 1 (A–C), Fig. 4 B, Fig. 5, and Fig. 6 D were obtained as follows. All images were collected on a microscope. The type, magnifi cation, and NA of the objective lenses were UPlanAPO, 60× oil, Materials and methods and NA 1.40, respectively. The experiments were performed at room temperature using Hoechst 33258 and FITC-labeled secondary antibodies C. elegans strains and culturing as fl uorochromes. Images were captured on a camera (model 2.1.1; The N2 Bristol strain was used as wild type in all control experiments and Diagnostic Instruments) and processed using SPOT Advanced version for all RNAi experiments. SP482 (rad-3[mn15]), SP488 (rad-2[mn156]), 3.2.4 software (Diagnostic Instruments). TG5 (rad-51[lg8701]), VC531 (rad-54[ok615]), and VC18 (ubc-1[gk14]) strains were obtained from the Caenorhabditis Genetics Center. Animals Online supplemental material were maintained as described previously (Brenner, 1974). Fig. S1 shows that F10G8.7 and gei-17 embryos are sensitive to UV light. The supplemental text describes UV sensitivity assays and the observation that RNAi gei-17 RNAi causes in UV sensitivity in embr yos. Online supplemental material is RNAi by feeding was performed for F10G8.7, W03A3.2, Y37B11A.2, available at http://www.jcb.org/cgi/content/full/jcb.200512136/DC1. ZK675.2, atl-1, gei-17, and chk-1 as described previously (Timmons and Fire, 1998). All bacteria were cultured for 24 h at 37°C in Terrifi c Broth We dedicate this work to John Newport, a pioneer in the study of early containing 50 μg/ml ampicillin, seeded onto nematode growth media embryonic cell cycles. (Brenner, 1974) plates containing 5 mM IPTG, and allowed to dry over- We thank Craig Hunter and Tim Schedl for advice. night. With the exception of chk-1 RNAi, worms were grown for two gen- Some of the strains used in this work were provided by the Caenorhabditis erations on RNAi bacteria. F1 progeny of chk-1 RNAi worms are sterile; Genetics Center, which is funded by the National Institutes of Health therefore, chk-1 RNAi was fed for one generation and analysis was per- National Center for Research Resources. A. La Volpe was supported by a DAMAGE CHECKPOINT SILENCING • HOLWAY ET AL. 1007 MacQueen, A.J., and A.M. Villeneuve. 2001. Nuclear reorganization and homol- Telethon-Italy grant (GGP04010). A.H. Holway was supported by a National ogous chromosome pairing during meiotic prophase require C. elegans Institute of General Medical Sciences (NIGMS) Genetics and Genomics training chk-2. 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Lupardus, P.J., T. Byun, M.C. Yee, M. Hekmat-Nejad, and K.A. Cimprich. 2002. A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev. 16:2327–2332. 1008 JCB • VOLUME 172 • NUMBER 7 • 2006

Journal

The Journal of Cell Biology – Pubmed Central

Published: Mar 27, 2006

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