Claspin, a Chk1-Regulatory Protein, Monitors DNA Replication on Chromatin Independently of RPA, ATR, and Rad17

Molecular Cell, Vol. 11, 329–340, February, 2003, Copyright 2003 by Cell Press

Claspin, a Chk1-Regulatory Protein, Monitors DNA Replication on Chromatin Independently of RPA, ATR, and Rad17 Joon Lee, Akiko Kumagai, and William G. Dunphy* Division of Biology 216-76 Howard Hughes Medical Institute California Institute of Technology Pasadena, California 91125

Summary Claspin is required for the ATR-dependent activation of Chk1 in Xenopus egg extracts containing incompletely replicated DNA. We show here that Claspin associates with chromatin in a regulated manner during S phase. Binding of Claspin to chromatin depends on the pre-replication complex (pre-RC) and Cdc45 but not on replication protein A (RPA). These dependencies suggest that binding of Claspin occurs around the time of initial DNA unwinding at replication origins. By contrast, both ATR and Rad17 require RPA for association with DNA. Claspin, ATR, and Rad17 all bind to chromatin independently. These findings suggest that Claspin plays a role in monitoring DNA replication during S phase. Claspin, ATR, and Rad17 may collaborate in checkpoint regulation by detecting different aspects of a DNA replication fork. Introduction Eukaryotic cells utilize checkpoint control mechanisms to safeguard the integrity of their genomes. For example, these regulatory pathways prevent entry into mitosis if DNA replication has not been completed normally or if the genome has suffered various types of DNA damage (O’Connell et al., 2000; Melo and Toczyski, 2002). These networks contain sensor proteins that interact directly or indirectly with various DNA structures at replication forks or sites of damage. These sensor proteins control downstream effector kinases, which in turn regulate cell cycle progression and other processes. Genetic studies in yeast have identified many components of these checkpoint systems. In fission yeast, the sensor proteins include Rad1, Rad3, Rad9, Rad17, Rad26, and Hus1 (O’Connell et al., 2000). Chk1 and Cds1 function as effector kinases in this system. Rad3 is a kinase in the phosphatidylinositol kinase (PIK)-related family that regulates the activation of Chk1 and Cds1 in response to various checkpoint-inducing DNA structures. In budding yeast, Mec1, a close relative of fission yeast Rad3, controls the effector kinases Chk1 and Rad53, a homolog of Cds1 (Melo and Toczyski, 2002). In vertebrates, the PIK relatives include ATM and ATR (Abraham, 2001). ATM phosphorylates various proteins after exposure to ionizing radiation, including Chk2/ Cds1 (Bartek et al., 2001). By contrast, ATR is essential for the phosphorylation-dependent activation of Chk1 in response to incompletely replicated DNA (Guo et al., 2000; Hekmat-Nejad et al., 2000; Liu et al., 2000; Zhao *Correspondence: [email protected]

and Piwnica-Worms, 2001). Chk1 inhibits the entry into mitosis by downregulating Cdc25 and upregulating Wee1, which together control the timing of the G2/M transition (O’Connell et al., 2000; Melo and Toczyski, 2002). We have been using Xenopus egg extracts to study the checkpoint-regulatory pathway that monitors the progression of S phase. In these extracts, Xenopus Chk1 (Xchk1) has a key role in assessing the successful completion of DNA replication (Kumagai et al., 1998; Guo et al., 2000; Kumagai and Dunphy, 2000; Lee et al., 2001). Xchk1 becomes hyperphosphorylated and undergoes a substantial increase in kinase activity in egg extracts containing DNA replication inhibitors such as aphidicolin (Kumagai and Dunphy, 2000). Aphidicolin, which inhibits polymerase ␣ (Pol ␣) and other replicative polymerases, causes the formation of stalled replication forks with primed DNA templates (Michael et al., 2000). Certain synthetic oligonucleotides also trigger the activation of Xchk1. For example, an annealed mixture of poly(dA)70 and poly(dT)70 elicits the activation of Xchk1 efficiently (Kumagai and Dunphy, 2000). The activation of Xchk1 is dependent on the Xenopus homolog of ATR (Xatr), which phosphorylates Xchk1 on four Ser-Gln/Thr-Gln (SQ/TQ) motifs in its C-terminal domain (Guo et al., 2000). A key issue is how cells detect incompletely replicated DNA by using checkpoint proteins. In all eukaryotes, an ordered sequence of events leads to the initiation of DNA replication (Bell and Dutta, 2002). The origin recognition complex (ORC) recruits Cdc6, Cdt1, and Mcm2-7 to origins to form a pre-replication complex (pre-RC). At the beginning of S phase, the protein kinases Cdc7 and Cdk2 collaborate to incorporate Cdc45 into the pre-RC, which results in formation of the preinitiation complex (pre-IC) (Mimura and Takisawa, 1998; Zou and Stillman, 1998; Jares and Blow, 2000; Walter and Newport, 2000; Walter, 2000). Thereafter, origin unwinding leads to binding of RPA and loading of various polymerases onto the DNA. Initially, Pol ␣ synthesizes short RNA-DNA primers on this unwound structure. Next, replication factor C (RFC), a complex of five proteins (RFC1-5), loads the trimeric PCNA protein onto these primed templates. PCNA is a sliding clamp that acts as a processivity factor for polymerase delta (Pol ␦). Significantly, Rad17 is closely related to the large subunit of RFC (Griffiths et al., 1995). In addition, Rad9, Rad1, and Hus1 form a trimer (the 9-1-1 complex) that may function as a PCNA-like clamp (Venclovas and Thelen, 2000). Various studies have indicated that a complex of Rad17 and RFC2-5 loads the 9-1-1 complex onto chromatin (Melo and Toczyski, 2002). Recent findings in various experimental systems have established that ATR associates with chromatin independently of Rad17 and the 9-1-1 complex, which indicates that ATR and these proteins recognize distinct DNA structures (Kondo et al., 2001; Melo et al., 2001; You et al., 2002; Zou et al., 2002). Our laboratory has identified Claspin as a conserved protein in vertebrates that is essential for activation of

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Chk1 (Kumagai and Dunphy, 2000). Claspin associates with Xchk1 in Xenopus egg extracts during a DNA replication checkpoint response. Furthermore, in the absence of Claspin, Xchk1 cannot undergo activation and thus cannot trigger a cell cycle delay in the presence of incompletely replicated DNA. Therefore, Claspin is necessary for the Xatr-dependent phosphorylation of Xchk1. Potential analogs of Claspin called Mrc1 have been identified in both budding and fission yeast (Alcasabas et al., 2001; Tanaka and Russell, 2001). Claspin also shares functional properties with budding yeast Rad9 and fission yeast Crb2 (O’Connell et al., 2000; Melo and Toczyski, 2002). An important question is whether Claspin and its analogs interact with chromatin during the execution of their functions. If so, do these proteins participate with other checkpoint proteins in sensing DNA structures, or do they act solely downstream of the sensor proteins to transmit information to effector kinases? In this report, we present evidence that Claspin monitors the process of DNA replication as part of its function. Results Claspin Binds to Replicating Chromatin Previously, we demonstrated that Claspin undergoes phosphorylation in Xenopus egg extracts containing certain synthetic oligonucleotides, such as a poly(dA)70poly(dT)70 (Kumagai and Dunphy, 2000). We asked whether Claspin becomes similarly modified in extracts containing incompletely replicated sperm chromatin. For this purpose, we incubated demembranated frog sperm chromatin in egg extracts in the absence and presence of aphidicolin. We observed by immunoblotting that Claspin became modified in aphidicolin-containing extracts, as shown by a reduced electrophoretic mobility (Figure 1A). This modification was reversed by treatment with lambda protein phosphatase (data not shown). Furthermore, the aphidicolin-dependent phosphorylation of Claspin was abolished in the presence of caffeine, an inhibitor of ATR and ATM. To evaluate whether this phosphorylation requires semi-conservative DNA synthesis, we used two inhibitors of ORC-mediated replication. Geminin inhibits formation of the pre-RC by blocking the loading of the Mcm proteins (McGarry and Kirschner, 1998). In addition, p21-N, the Cdk-inhibitory domain of p21, prevents incorporation of Cdc45 into the pre-IC by inhibiting Cdk2-cyclin E (Jares and Blow, 2000; Walter, 2000). We found that both Geminin and p21-N abolished the modification of Claspin (Figure 1A). Under these conditions, both Geminin and p21-N also prevented the checkpointdependent phosphorylation of Xchk1, as judged by its electrophoretic mobility and reactivity with anti-PSer344 antibodies (Figure 1A). Overall, these findings indicate that Claspin undergoes phosphorylation in response to DNA replication in egg extracts. Next, we examined whether Claspin could associate with chromatin during S phase. Initially, we added sperm chromatin to the egg extracts, reisolated the chromatin at various times, and then probed for Claspin by immunoblotting. In egg extracts, nuclear envelope assembly around exogenously added sperm chromatin occurs in

30–40 min. Once the membrane seals off the nuclear interior from the cytoplasm, initiation of DNA replication commences shortly thereafter. We observed that Claspin bound to chromatin in a time-dependent manner (Figure 1B). Typically, the binding of Claspin began at around 40 min, peaked near 60 min, and declined subsequently. The initial binding of pre-RC components such as Xorc2 and Xmcm7 preceded the association of Claspin with chromatin. The binding profiles of several later-acting replication proteins (e.g., RPA, Pol ␣, Pol ⑀, and PCNA) were similar but not identical to that of Claspin. In parallel, we examined the effect of aphidicolin on the binding of Claspin (Figure 1B). Treatment with aphidicolin resulted in elevated and prolonged binding of Claspin. Typically, by 90 to 120 min, Claspin accumulated to high levels on aphidicolin-treated chromatin. By this time, the aphidicolin-induced phosphorylation of Xchk1 had reached its maximum (Figure 1C). There was also increased binding of Claspin to UV-damaged sperm chromatin (Figure 1D), which would likewise accumulate DNA replication blocks (Kumagai et al., 1998). As expected, treatment with aphidicolin led to increased levels of RPA70 and Pol ␣ on chromatin (Walter and Newport, 2000). Conversely, there was less PCNA bound under these conditions. We also compared the behavior of other Xenopus checkpoint proteins with that of Claspin (Figure 1B). In the absence of aphidicolin, Xatr, Xrad17, and Xhus1 all bound to chromatin at around 40 min and remained associated in significant amounts for up to 120 min. In the presence of aphidicolin, Xatr, Xrad17, and Xhus1 all accumulated to high levels on chromatin like Claspin. Taken together, these findings demonstrate that Claspin binds to replicating chromatin during S phase. To examine whether this binding involves the route used by DNA replication proteins, we utilized Geminin and p21-N. We observed that Geminin and p21-N each completely blocked the binding of Claspin in both the absence and presence of aphidicolin (Figure 1E). In control experiments, we observed that Geminin also prevented the binding of both Xmcm7 and RPA, whereas p21-N inhibited the association of RPA but not Xmcm7 with chromatin. Thus, the binding of Claspin to chromatin occurs by a mechanism that requires both a functional pre-RC and Cdk2. Claspin Depends Upon Xcdc45 for Binding to Chromatin We next sought to delineate more precisely the requirements for binding of Claspin to chromatin. It is currently thought that Cdc45 promotes the initial unwinding of DNA at replication origins (Mimura and Takisawa, 1998; Zou and Stillman, 1998; Mimura et al., 2000; Walter and Newport, 2000). The binding of Cdc45 to chromatin requires both the pre-RC and Cdk2. Consequently, Cdk2 inhibitors such as p21 block the association of Cdc45 with potential sites of replication (Jares and Blow, 2000; Walter and Newport, 2000). Since the binding of Claspin to chromatin also requires Cdk2, we directly examined the role of Cdc45 in this binding. For these experiments, we used polyclonal antibodies to deplete Xenopus Cdc45 (Xcdc45) from egg extracts. As shown in Figure 2, extracts treated with these antibodies contained neg-

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ligible Xcdc45 in comparison with extracts that had been mock-depleted with nonspecific antibodies. We incubated sperm chromatin in the antibody-treated extracts in the absence and presence of aphidicolin. Subsequently, we isolated chromatin fractions from reconstituted nuclei that had formed during the incubation and probed these fractions with antibodies to Claspin. We also used antibodies against Xcdc45 to assess its level on chromatin. In addition, we analyzed the chromatin fractions with antibodies to Pol ⑀ and RPA70. It is known that binding of both Pol ⑀ and RPA to chromatin requires Xcdc45 (Mimura et al., 2000). However, Pol ⑀ does not need RPA for initial association with chromatin (Mimura et al., 2000). Thus, Pol ⑀ and RPA represent markers for distinct loading events that are both downstream of the Xcdc45-dependent step. We found that there was negligible binding of Claspin to Xcdc45-depleted chromatin in both the absence and presence of aphidicolin (Figure 2). Similarly, there was little or no binding of Pol ⑀ and RPA in the absence of Xcdc45. We have not been able to coimmunoprecipitate Xcdc45 and Claspin from egg extracts, suggesting that these proteins do not load onto chromatin as a complex (data not shown). Finally, we found that addition of recombinant Xcdc45 efficiently restored the association of Claspin, Pol ⑀, and RPA with chromatin. From these experiments, we conclude that the loading of Claspin (as well as that of Pol ⑀ and RPA) onto prospective sites of replication requires the presence of Xcdc45. In timecourse experiments, we observed that Xcdc45, Claspin, RPA, and PCNA all associated with DNA within a 3 min interval approximately 40 min after addition of sperm chromatin (data not shown). Thus, once Xcdc45 associates with chromatin, the binding of Claspin and proteins involved in the initiation and elongation phases of replication occurs rapidly.

Figure 1. Claspin Is a Chromatin Binding Protein (A) Phosphorylation of Claspin and Xchk1 in aphidicolin-treated extracts. Sperm chromatin was incubated for 90 min in egg extracts in the absence (lane 1) or presence (lanes 2–5) of 100 ␮g/ml aphidicolin. In some cases, 5 mM caffeine (lane 3) or 1 ␮M p21-N (lane 5) was also present. For lane 4, the extract was pretreated for 20 min on ice with 300 nM Geminin. After incubation, the extracts were analyzed by immunoblotting with anti-Claspin antibodies (top). Nuclear fractions from the extracts were immunoblotted with antibodies that detect either the whole Xchk1 protein (middle) or P-Ser344 of Xchk1 (bottom). APH, aphidicolin. (B) Claspin associates with replicating chromatin. Sperm chromatin was incubated in egg extracts in the absence (lanes 1–5) or presence (lanes 6–10) of aphidicolin. At the times shown, chromatin fractions were isolated from the extracts and analyzed for the indicated proteins by immunoblotting. (C) Time-course for phosphorylation of Xchk1. Sperm chromatin was incubated in egg extracts in the absence (lanes 1–4) or presence

Recruitment of Claspin to Chromatin Does Not Require RPA RPA is a heterotrimeric, single-stranded DNA binding protein that is crucial for various aspects of DNA metabolism (Wold, 1997). For DNA replication, RPA stabilizes unwound single-stranded DNA at origins and thereby creates a template for Pol ␣ (Mimura et al., 2000; Walter and Newport, 2000). To examine the relationship between Claspin and RPA, we carried out immunodepletion of RPA from egg extracts with antibodies against its 70 kd subunit (RPA70). As shown by immunoblotting, RPA70 could be removed completely from egg extracts

(lanes 5–8) of aphidicolin. At the times shown, nuclear fractions were isolated and probed by immunoblotting with antibodies that detect the whole Xchk1 protein (top) or P-Ser344 of Xchk1 (bottom). (D) Undamaged (lane 1) and UV-damaged (lane 2) sperm chromatin were incubated in extracts for 90 min. Binding of Xatr, Claspin, and Xorc2 to chromatin was determined by immunoblotting. (E) Binding of Claspin to chromatin requires the pre-RC and Cdk2. Extracts were treated with buffer alone (lanes 1 and 4), Geminin (lanes 2 and 5), or p21-N (lanes 3 and 6) as in (A). Sperm chromatin was incubated in the extracts in the absence of aphidicolin for 60 min (lanes 1–3) or the presence of aphidicolin for 90 min (lanes 4–6). Chromatin fractions were isolated and probed for the indicated proteins by immunoblotting.

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Figure 2. Xcdc45 Is Required for Loading of Claspin onto Chromatin Egg extracts were treated with control (lanes 1, 4, and 5) or anti-Xcdc45 antibodies (lanes 2, 3, and 6–9). Recombinant Xcdc45 (rXcdc45) was added back after the immunodepletion (lanes 3, 8, and 9). Sperm chromatin was incubated in the various extracts for 90 min in the absence (lanes 4, 6, and 8) or presence of aphidicolin (lanes 5, 7, and 9) and chromatin fractions were isolated. The indicated proteins were detected by immunoblotting. ⌬Mock and ⌬Xcdc45 refer to mock-depleted and Xcdc45-depleted extracts.

with these antibodies (Figure 3A). To verify the effectiveness of the immunodepletion procedure, we assayed chromosomal DNA replication in the RPA70-depleted extracts. As shown in Figure 3B, DNA replication was strongly compromised in RPA70-depleted extracts. DNA synthesis could be restored by the addition of recombinant human RPA. We proceeded to examine the binding of Claspin to chromatin in RPA-depleted extracts (Figure 3C). We found that Claspin bound very well to RPA-depleted chromatin in both the absence and presence of aphidicolin. Indeed, in both cases, the binding was substantially higher than for chromatin from mock-depleted extracts that contained aphidicolin. As expected, there was negligible Pol ␣ or PCNA on RPA-depleted chromatin in the absence or presence of aphidicolin. As shown in Figure 3D, addition of recombinant RPA to RPAdepleted extracts restored the behavior of Claspin to that seen in mock-depleted extracts (i.e., there was strongly increased binding in the presence of aphidicolin). Likewise, recombinant RPA restored the binding of Pol ␣ to chromatin. These observations clearly indicate that Claspin does not need RPA to associate with chromatin. Furthermore, the lack of RPA causes some sort of deregulation that results in elevated binding of Claspin in both the absence and presence of aphidicolin. We also compared the binding characteristics of Xmcm7 and Pol ⑀ with those of Claspin in the absence of RPA (Figure 3C). Like Claspin, both Xmcm7 and Pol ⑀ bound equally well to RPA-depleted chromatin in the absence or presence of aphidicolin. As described earlier, Claspin, in contrast to Xmcm7, requires Xcdc45 for binding to chromatin. Claspin and Pol ⑀ have similar properties in that both proteins require Xcdc45 but not RPA in order to associate with chromatin. To assess whether the increased binding of Claspin to RPA-depleted chromatin also requires the pre-RC, we utilized Geminin. For this experiment, we incubated RPA-depleted extracts in the absence or presence of Geminin and subsequently probed chromatin fractions with anti-Claspin antibodies. We observed that Geminin

completely blocked the binding of Claspin to RPAdepleted chromatin (Figure 3E). In this experiment, Geminin also abolished the binding of both Xmcm7 and Pol ⑀ to chromatin. These findings indicate that the elevated binding of Claspin that occurs in the absence of RPA still requires a functional pre-RC. Binding of Xatr to Single-Stranded Regions of DNA Requires RPA Recently, it was shown that association of Xatr with chromatin in aphidicolin-treated Xenopus egg extracts requires RPA (You et al., 2002). In our experiments, we observed that there is a low amount of Xatr on chromatin in the absence of aphidicolin and that binding of Xatr increases substantially following treatment with aphidicolin (Figure 3C). In the absence of RPA, we observed a low amount of Xatr on chromatin in both the absence and presence of aphidicolin. As described above, Claspin accumulates to high levels on RPA-depleted chromatin under both conditions. In order to characterize the RPA-dependent binding of Xatr to chromatin, we used a defined single-stranded DNA template. In particular, we examined the binding of Xatr to the DNA homopolymer poly(dA)70 in the absence and presence of RPA (Figure 3F). This DNA template is not capable of undergoing replication to a double-stranded form in egg extracts (Guo and Dunphy, 2000; Kumagai and Dunphy, 2000). Furthermore, poly(dA)70 does not trigger the activation of Xchk1 in these extracts (Kumagai and Dunphy, 2000). As shown in Figure 3F, Xatr binds very well to magnetic beads that contain poly(dA)70, but not to control beads that lack DNA. We found that the binding of Xatr to poly(dA)70 was abolished in the absence of RPA. Moreover, recombinant human RPA efficiently restored the interaction of Xatr with this single-stranded DNA template. These experiments argue that tight association of Xatr with single-stranded regions of DNA requires the presence of RPA. Since poly(dA)70 cannot trigger the activation of Xchk1, the Xatr that is bound to this template is presumably incapable of phosphorylating Xchk1. Notably, there is no binding of Xmcm7 and

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Figure 3. RPA Is Not Required for Interaction of Claspin with Chromatin (A) Immunodepletion of RPA. Extracts were treated with control (lane 1) or anti-Xenopus RPA70 antibodies (lanes 2 and 3). For lane 3, recombinant human RPA (rhRPA) was added back to the depleted extracts at a final concentration of 24 ␮g/ml. The amounts of RPA70, Xatr, and Claspin in the extracts were determined by immunoblotting. These anti-RPA70 antibodies do not crossreact with human RPA70. (B) DNA replication in RPA-depleted extracts. DNA replication was measured in the extracts from (A) as described (Coleman et al., 1996). (C) Binding of Claspin to chromatin lacking RPA. Sperm chromatin was incubated in mock-depleted (lanes 1 and 2) or RPA-depleted extracts (lanes 3 and 4) for 90 min in the absence (lanes 1 and 3) or presence of aphidicolin (lanes 2 and 4). Chromatin fractions were isolated and probed for the indicated proteins by immunoblotting. (D) Addition of rhRPA to RPA-depleted extracts restores normally regulated binding of Claspin and Pol ␣ to chromatin. Sperm chromatin was incubated for 90 min in RPA-depleted extracts containing rhRPA in the absence (lane 1) or presence (lane 2) of aphidicolin. Chromatin fractions were isolated, and probed for Claspin and Pol ␣ by immunoblotting. (E) Binding of Claspin to chromatin in RPA-depleted extracts requires the pre-RC. Sperm chromatin was incubated for 90 min in mockdepleted extracts (lane 1), RPA-depleted extracts (lane 2), or RPA-depleted extracts that had been treated with Geminin (lane 3). Chromatin fractions were isolated and binding of Claspin, Xmcm7, and Pol ⑀ was determined by immunoblotting. (F) RPA is necessary for binding of Xatr to single-stranded DNA. Magnetic beads containing no DNA (lane 2) or poly(dA)70 (lanes 3–5) were incubated for 60 min in mock-depleted extract (lanes 2 and 3), RPA-depleted extract (lane 4), or RPA-depleted extract containing rhRPA (lane 5). The beads were isolated and washed. RPA, Xatr, Xmcm7, and Claspin were detected by immunoblotting. Lane 1 shows untreated whole egg extract (1 ␮l).

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Claspin to poly(dA)70 in the absence or presence of RPA (Figure 3F). Thus, unlike Xatr, Claspin cannot interact with single-stranded DNA that is not associated with a DNA replication fork. Removal of Claspin Does Not Prevent the Binding of Other Checkpoint Proteins to Chromatin Because Claspin associates with DNA at an early stage in the process of replication, we asked whether Claspin has any role in controlling the binding of DNA replication proteins and other checkpoint regulatory proteins to chromatin (Figure 4). For these experiments, we removed Claspin from egg extracts with affinity-purified antibodies. To verify the efficacy of the immunodepletion, we confirmed that the checkpoint-dependent phosphorylation of Xchk1 was abolished in these experiments (Figures 4A and 4C). Previously, we reported that Claspin-depleted extracts are proficient for DNA replication (Kumagai and Dunphy, 2000). As shown in the time-course experiment in Figure 4B, the extent of DNA replication in mockdepleted and Claspin-depleted extracts is quite similar, although there is a modest slowing of replication in the absence of Claspin. As another means to characterize DNA replication in the absence of Claspin, we analyzed the binding of various key replication proteins to Claspin-depleted chromatin. We observed that Xorc2, Xmcm7, RPA70, and Pol ␣ all bound normally to chromatin that lacks Claspin (Figure 4C). We turned to the question of whether Claspin would affect the ability of other checkpoint proteins to associate with chromatin. Initially, we focused on the relationship between Claspin and Xatr. As described above, Claspin does not require RPA to associate with chromatin. Conversely, the elevated binding of Xatr to chromatin in aphidicolin-treated extracts depends upon RPA. These observations suggest that recruitment of Xatr to sites of replication occurs downstream of the Claspin binding step. To ask if Xatr depends on Claspin for binding to chromatin, we examined the amount of Xatr in chromatin fractions from Claspin-depleted extracts. As shown in Figure 4C, there was no difference in the amount of chromatin-bound Xatr in mock-depleted versus Claspin-depleted extracts in the absence or presence of aphidicolin. Thus, Claspin is not necessary for recruitment of Xatr to chromatin. We also investigated the binding of Xrad17 and Xhus1 to chromatin in the absence of Claspin. We found that both Xrad17 and Xhus1 could bind to chromatin in Claspin-depleted, aphidicolin-treated extracts (Figure 4C). Interestingly, we consistently observed elevated binding of Xrad17 but not Xhus1 to chromatin in the absence of Claspin. To characterize this effect further, we compared the binding of Xrad17 and Xhus1 to chromatin in the absence of RPA or Claspin or both (Figure 4D). We observed that neither Xrad17 nor Xhus1 could bind to RPA-depleted chromatin in aphidicolin-treated extracts. Binding of both Xrad17 and Xhus1 could be restored by the addition of recombinant human RPA (data not shown). You et al. (2002) have also reported that binding of Xhus1 to chromatin depends upon RPA. As described above, there was increased binding of Xrad17 but not Xhus1 to Claspin-depleted chromatin.

By contrast, there was no binding of either Xrad17 or Xhus1 to chromatin in extracts that lacked both Claspin and RPA, indicating that the elevated binding of Xrad17 in the absence of Claspin still depends on RPA. Taken together, these observations indicate that the absence of Claspin does not compromise the binding of DNA replication factors to chromatin, which is consistent with the fact that DNA synthesis occurs efficiently in Claspin-depleted extracts. Furthermore, Claspin is not needed for the interaction of other checkpoint factors (e.g., Xatr, Xrad17, and Xhus1) with chromatin. However, Claspin appears to regulate Xrad17 on DNA in some manner because Xrad17 accumulates to higher levels on aphidicolin-treated chromatin in the absence of Claspin. Xatr and Xrad17 Regulate the Amount of Claspin on Chromatin ATR and Rad17 have been implicated in the regulation of Chk1 in various organisms (Melo and Toczyski, 2002). Thus, we asked whether Xatr and Xrad17 would affect the binding of Claspin to chromatin. In Xenopus egg extracts, the phosphorylation-dependent activation of Xchk1 is absolutely dependent on both Xatr and Claspin (Guo et al., 2000; Kumagai and Dunphy, 2000). However, the role of Xrad17 in the regulation of Xchk1 has not been characterized previously. Therefore, in conjunction with the chromatin binding studies, we examined whether Xrad17 is involved in the activation of Xchk1 in egg extracts. We used affinity-purified antibodies to remove Xrad17 from egg extracts (Figure 5A). In parallel, we also immunodepleted Xatr from the extracts (Figure 5A). Next, we added sperm chromatin to the antibody-treated extracts in the absence and presence of aphidicolin. After a 90 min incubation, we examined phosphorylation of Xchk1 by immunoblotting with antibodies that detect phosphorylation of Xchk1 on Ser344. Consistent with previous results (Guo et al., 2000), there was no phosphorylation of Ser344 in Xatr-depleted extracts (Figure 5B). In Xrad17-depleted extracts, we consistently observed that the phosphorylation of Xchk1 was substantially reduced but not completely abolished (Figure 5B). The phosphorylation of Xchk1 on Ser344 in Xrad17-depleted extracts was 22.0 ⫾ 8.7% (average ⫾ SD from three experiments) of that observed in mock-depleted extracts. It is unlikely that the remaining phosphorylation of Xchk1 is due to incomplete removal of Xrad17 because there is no nuclear uptake of Xhus1 and, consequently, no binding of Xhus1 to chromatin in these Xrad17depleted extracts (Figures 5B and 5D). It is well established that Rad17 is required for nuclear accumulation of Hus1 in fission yeast (Caspari et al., 2000). It was important to analyze DNA replication in the Xrad17-depleted and Xatr-depleted extracts. As shown in Figure 5C, DNA replication occurred efficiently in the absence of Xrad17 or Xatr. In other systems, Rad17 exists in a complex with the four small subunits of RFC, which is essential for DNA replication (Uhlmann et al., 1996). By immunoblotting, we found that immunodepletion of Xrad17 did not substantially reduce the supply of RFC36, RFC37, RFC38, and RFC40 in the extracts (data not shown). Overall, these experiments indicate

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Figure 4. Claspin Is Not Required for Binding of DNA Replication Proteins or Other Checkpoint Proteins to Chromatin (A) Phosphorylation of Xchk1 in the absence and presence of Claspin. Extracts were treated with control antibodies (lanes 1 and 2) or antiClaspin antibodies (lane 3). Sperm chromatin was incubated in the egg extracts in the absence (lane 1) or presence (lanes 2 and 3) of aphidicolin. Nuclear fractions were isolated and probed by immunoblotting with antibodies against the whole Xchk1 protein (top) or P-Ser344 of Xchk1 (bottom). (B) DNA replication in Claspin-depleted extracts. (C) Binding of DNA replication proteins and other checkpoint proteins to chromatin in the absence of Claspin. Mock-depleted (lane 1) and Claspin-depleted extracts (lane 2) were prepared. Sperm chromatin was incubated for 90 min in mock-depleted (lanes 3 and 4) and Claspindepleted extracts (lanes 5 and 6) in the absence (lanes 3 and 5) or presence (lanes 4 and 6) of aphidicolin. After 90 min, chromatin fractions were isolated. The extracts and chromatin fractions were probed for the indicated proteins by immunoblotting. (D) Roles of Claspin and RPA in the binding of Xrad17 and Xhus1 to chromatin. Extracts were treated with control antibodies (lanes 1, 5, and 6), anti-Claspin antibodies (lanes 2 and 7), anti-RPA antibodies (lanes 3 and 8), or both anti-Claspin and anti-RPA antibodies (lanes 4 and 9). Sperm chromatin was added to extracts lacking (lane 5) or containing (lanes 6–9) aphidicolin. After 90 min, chromatin fractions were isolated (lanes 5–9). The amounts of Claspin, RPA, Xrad17, and Xhus1 in the extracts and chromatin fractions were determined by immunoblotting.

that Xrad17 contributes to the maximal activation of Xchk1, but there appears to be a Rad17-independent process that results in significant phosphorylation of Xchk1 in the absence of Xrad17. In this respect, Xrad17

differs from Xatr and Claspin, both of which are absolutely required for the activation of Xchk1. Next, we examined the amount of Claspin on chromatin in both Xatr-depleted and Xrad17-depleted extracts

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Figure 5. Binding of Claspin to Chromatin Is Increased in the Absence of Xatr or Xrad17 (A) Immunodepletion of Xatr and Xrad17. Egg extracts were treated with control antibodies (lane 1), anti-Xatr antibodies (lane 2), or antiXrad17 antibodies (lane 3). The amounts of Xatr, Xrad17, and Xhus1 in the extracts were determined by immunoblotting. (B) Phosphorylation of Xchk1 in extracts lacking Xatr or Xrad17. Sperm chromatin was incubated in the extracts from (A) in the absence (lane 1) or presence (lanes 2–4) of aphidicolin. Nuclear fractions were isolated from the extracts and were probed by immunoblotting with antibodies against Xchk1, P-Ser344 of Xchk1, Xrad17, Xhus1, and PCNA. (C) DNA replication in the absence of Xatr and Xrad17. (D) Binding of proteins to chromatin in the absence of Xatr or Xrad17. Mock-depleted (lanes 1 and 2), Xatr-depleted (lanes 3 and 4), and Xrad17-depleted (lanes 5 and 6) extracts were prepared. Sperm chromatin was incubated in the various extracts in the absence (lanes 1, 3, and 5) or presence (lanes 2, 4, and 6) of aphidicolin. After 90 min, chromatin fractions were isolated from the extracts and analyzed for the indicated proteins by immunoblotting. (E) Effect of caffeine on the binding of checkpoint and replication proteins to chromatin. Extracts were incubated with sperm chromatin for 90 min in the presence of no drug (lane 2), aphidicolin (lane 3), or both aphidicolin and caffeine (lane 4). After 90 min, chromatin fractions were isolated. The initial extract (1 ␮l, lane 1) and chromatin fractions (lanes 2–4) were probed for the indicated proteins by immunoblotting.

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in the absence and presence of aphidicolin. We observed that Claspin clearly does not need either Xatr or Xrad17 to associate with chromatin (Figure 5D). Indeed, Claspin accumulated to much higher levels on aphidicolin-treated chromatin in both Xatr-depleted and Xrad17-depleted extracts in comparison with mockdepleted extracts. In addition, there were elevated levels of both Xrad17 and Xhus1 on chromatin in Xatr-depleted extracts containing aphidicolin. In the absence of Xrad17, Xatr bound well to chromatin but, as expected, there was no binding of Xhus1. These findings are consistent with observations in other systems that ATR and Rad17 bind to chromatin independently of one another (Kondo et al., 2001; Melo et al., 2001; Zou et al., 2002). From our studies, we can conclude that Claspin associates with chromatin independently of both ATR and Rad17. Thus, Claspin, Xatr, and Xrad17 presumably interact with distinct nucleic acid structures or proteins or both at sites of replication. As part of these experiments, we also analyzed the binding of various DNA replication factors to chromatin in the absence of Xatr or Xrad17 (Figure 5D). We found that binding of two components of the pre-RC, namely Xorc2 and Xmcm7, was unaffected by the removal of Xatr or Xrad17. Conversely, Pol ⑀, RPA, and Pol ␣ accumulated on aphidicolin-treated chromatin at much higher levels in Xatr-depleted and Xrad17-depleted extracts than in mock-depleted extracts. As another means to analyze these findings, we used caffeine, an inhibitor of ATR and ATM (Abraham, 2001). We observed that treatment with both aphidicolin and caffeine resulted in a large increase of Xatr, Claspin, Xrad17, Xhus1, Pol ⑀, RPA, and Pol ␣ on chromatin (Figure 5E). Caffeine alone did not affect binding of these proteins to chromatin (data not shown). By contrast, the binding of Xorc2 and Xmcm7 to DNA did not increase in the presence of both aphidicolin and caffeine. Thus, removal of Xatr by immunodepletion and inhibition of Xatr (and possibly Xatm) with caffeine lead to similar consequences. In particular, regulators of Xchk1 and certain replication proteins accumulate to very high levels during a replication arrest if checkpoint signaling is blocked. Overall, these observations suggest that the Xatrdependent signaling pathway regulates its own components (e.g., Claspin and Xrad17) and key DNA replication proteins on chromatin during S phase. Discussion In this report, we have investigated the role of Claspin in the checkpoint pathway that monitors the presence of incompletely replicated DNA. By using Xenopus egg extracts, we have found that Claspin binds to chromatin during S phase in a regulated manner (Figure 6). This binding depends on early steps in a DNA replication cycle that are necessary for the firing of replication origins, including assembly of both the pre-RC and preIC. We further analyzed the requirements for binding of Claspin to chromatin by removing critical replication proteins from egg extracts. We observed that Claspin could not bind to chromatin in extracts lacking Xcdc45. By contrast, removal of RPA does not prevent the binding of Claspin to chromatin. This finding is significant

Figure 6. Summary of the Requirements for Binding of Claspin to Chromatin

because RPA is essential for the recruitment of numerous proteins that are necessary for the initiation and elongation phases of DNA replication, including Pol ␣-primase, PCNA, and Pol ␦ (Mimura et al., 2000; Walter and Newport, 2000). Therefore, our results suggest that Claspin binds to chromatin even before the commencement of DNA synthesis. Relatively little is known about the molecular details of what occurs at DNA replication origins after the binding of Xcdc45 but before the recruitment of RPA. It is currently thought that Xcdc45 promotes the initial unwinding of DNA at origins, but its exact biochemical function is unknown (Mimura et al., 2000; Walter and Newport, 2000). Subsequently, more extensive unwinding results in the association of RPA with longer stretches of single-stranded DNA. According to our present level of understanding about the firing of replication origins, the binding of Claspin would occur around the time of the initial unwinding step. We have also investigated the relationship between Claspin and other checkpoint proteins (e.g., Xatr and Xrad17). Recent findings in various systems have indicated that ATR and Rad17 homologs associate with

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DNA independently of one another, suggesting that these proteins recognize different features of chromatin (Kondo et al., 2001; Melo et al., 2001; You et al., 2002; Zou et al., 2002). As we have shown here, Claspin clearly does not need either Xatr or Xrad17 to associate with chromatin. Thus, Claspin, Xatr, and Xrad17 each bind independently to chromatin during S phase. The implication is that these three proteins are involved in recognizing different facets of a DNA replication fork. Furthermore, we can conclude that Claspin most likely binds to replication sites before Xatr or Xrad17. ATR and Rad17 have been classified as checkpoint sensor proteins because they bind to DNA and participate in the activation of downstream effector kinases. By these criteria, Claspin can also now be regarded as a checkpoint sensor protein. An important question is what aspect of the DNA replication fork would serve as the signal that recruits Claspin. Claspin presumably interacts with some protein component(s) or DNA structure or both that would be present around the time of unwinding. Thus far, we have not been able to observe direct binding of Claspin to DNA (data not shown), which suggests that the association of Claspin with chromatin would be mediated by protein-protein interactions. Furthermore, Claspin cannot bind to defined, singlestranded DNA templates in egg extracts. The initial unwinding of DNA at origins is carried out by a helicase that would be dependent, directly or indirectly, on Xcdc45. It is widely suspected that the Mcm2-7 complex possesses a helicase activity (Bell and Dutta, 2002). This activity could be involved in the initial DNA unwinding step or the progression of replication forks or both. The unwinding of DNA and the accompanying topological strains are dynamic features of a DNA replication fork that could be monitored by Claspin in its role as a checkpoint sensor protein. Another possibility is suggested by the fact that Claspin and Pol ⑀ display similar dependencies for loading onto chromatin. More specifically, both Claspin and Pol ⑀ require Xcdc45 but not RPA for binding to chromatin. Mimura et al. (2000) originally reported the finding that Pol ⑀ does not need RPA to associate with chromatin in egg extracts. Consequently, binding of Pol ⑀ would have to occur before that of Pol ␣-primase. The exact role of Pol ⑀ in eukaryotic chromosomal replication is not known, but it is essential for normal DNA replication in Xenopus egg extracts (Waga et al., 2001). In Pol ⑀-depleted egg extracts, there is a severe reduction in overall DNA synthesis as well as accumulation of small replication intermediates. Pol ⑀ has been implicated in checkpoint responses in budding yeast (reviewed in Hu¨bscher et al., 2002). Overall, our observations raise the possibility that the functions of Claspin and Pol ⑀ may be interrelated. Various observations suggest that ATR recognizes single-stranded stretches of unwound DNA at replication forks. As shown in You et al. (2002) and in this paper, association of Xatr with chromatin in aphidicolin-treated extracts is severely reduced in the absence of RPA. Furthermore, we have observed that binding of Xatr to defined, single-stranded oligonucleotides such as poly(dA)70 is dependent upon RPA. This observation provides a strong argument that Xatr associates with RPA-

coated, single-stranded regions of DNA at replication forks. Significantly, single-stranded poly(dA)70 cannot trigger the activation of Xchk1 in egg extracts (Kumagai and Dunphy, 2000), even though Xatr binds very well to this template. By contrast, an annealed mixture consisting of poly(dA)70-poly(dT)70 elicits the activation of Xchk1 effectively (Kumagai and Dunphy, 2000). The implication is that this latter template and/or associated proteins trigger the activation of Xatr. Likewise, You et al. (2002) have presented evidence that Pol ␣ must synthesize primers for activation of Xchk1 to occur normally. By analogy with RFC1-5, a complex of Rad17 and RFC2-5 may load the 9-1-1 complex onto the ends of primed templates. Consistent with this possibility, binding of Xhus1 to chromatin is reduced in the absence of Pol ␣ (You et al., 2002). Taken together, these various observations argue that maximal activation of Xchk1 may involve recognition of three distinct features of DNA replication forks by checkpoint proteins. According to this scheme, Claspin would recognize some component that is associated with unwinding DNA. Secondly, Xatr would bind to RPA-containing, single-stranded regions. Finally, Xrad17 and Xhus1 may interact with primed templates. This triple-part mechanism would impart high specificity to the Claspin-dependent pathway. The loading of Claspin onto chromatin in a manner that depends on the pre-IC could also help to explain the organization of checkpoint signaling pathways. For the preservation of genomic integrity, the cell must monitor the fidelity of DNA replication, but it also must be able to cope with numerous types of damage that may occur in chromatin. ATR and Rad17 homologs have been implicated in both the DNA damage and DNA replication checkpoints in various organisms (Melo and Toczyski, 2002). These observations are consistent with the fact that ATR and Rad17 could interact with structures that would be found at both replication forks and sites of damage. Based on our observations, Claspin may be capable of interacting with chromatin only by a Cdc45mediated mechanism. By having both this chromatin binding specificity and the ability to interact with Xchk1, Claspin could direct Xatr and Xrad17 to the regulation of Xchk1 at sites of replication. Other factors like Claspin could interact with sites of DNA damage to facilitate distinct checkpoint responses. Although Claspin, Xatr, and Rad17 bind to chromatin independently, these proteins presumably collaborate with one another on the DNA. Both Claspin and Xatr are absolutely required for the checkpoint-dependent phosphorylation and activation of Xchk1. Furthermore, Xrad17 is necessary for maximal phosphorylation of Xchk1. By using anti-Claspin antibodies, we have been able to coimmunoprecipitate Claspin, Xatr, and Xrad17 from chromatin that has been extensively digested with micrococcal nuclease (data not shown). However, these three proteins cannot be coimmunoprecipitated from either whole egg extracts or from high-salt eluates of chromatin. Thus, it is unclear whether these proteins interact transiently on chromatin or simply bind to neighboring sites on DNA. In the human system, ATR can be found in a complex with Rad17 in cells that have been treated with hydroxyurea, ultraviolet light, and ionizing radiation (Bao et al., 2001).

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Through our immunodepletion studies, we have also obtained evidence that Xatr, Xrad17, and Claspin have some functional interrelationships on chromatin. For example, in both Xatr-depleted and Xrad17-depleted extracts, Claspin binds in strongly elevated amounts to aphidicolin-treated chromatin. Likewise, the amount of Xrad17 on chromatin increases greatly in the absence of Xatr. Previously, it has been shown that RPA and Pol ␣ accumulate to high levels on aphidicolin-treated chromatin in egg extracts. It was proposed that under these conditions DNA unwinding continues in the absence of DNA replication (Walter and Newport, 2000). As shown here, there is also increased binding of Claspin to aphidicolin-treated chromatin. The fact that binding of Claspin to aphidicolin-treated chromatin increases even further in the absence of Xatr and Xrad17 suggests that these proteins regulate Claspin in some manner. Rad17 has been identified as a substrate of ATR in humans (Bao et al., 2001), and we suspect that Claspin is a substrate of Xatr. Phosphorylation of Xrad17 and Claspin by Xatr could promote the dynamic cycling of these proteins between soluble and chromatin-bound forms. Another possibility is that Xatr suppresses the firing of new origins in aphidicolin-treated chromatin. Consequently, removal of Xatr (or Xrad17) would lead to the firing of additional origins and more binding of Claspin, RPA, and Pol ␣. In summary, we have found that Claspin interacts with chromatin in a regulated manner during the cell cycle. Moreover, Claspin senses the presence of a DNA replication fork in a manner that is distinct from that of either Xatr or Xrad17. This triple-part mechanism could enable the Claspin-dependent pathway to function with a high degree of accuracy. Experimental Procedures Recombinant Proteins Geminin (McGarry and Kirschner, 1998) and GST-p21-N (Chen et al., 1995) were prepared as described. Recombinant human RPA was kindly provided by M. Wold (Henricksen et al., 1994). Xcdc45His6 was generously supplied by J. Walter (Walter and Newport, 2000). Cloning of Xenopus Rad17 and Hus1 From conserved sequences in Rad17 homologs, two degenerate oligonucleotides were designed. A polymerase chain reaction (PCR) with these primers and Xenopus oocyte cDNA yielded a 600 bp fragment, which was used to isolate a 2.8 kb cDNA. Its open reading frame is 52% identical to human Rad17. A Xenopus Hus1 cDNA was isolated in a similar manner. Its sequence differs by only one codon from another Xhus1 clone (You et al., 2002). The GenBank accession numbers for Xrad17 and Xhus1 are AY169965 and AY169966, respectively. Antibodies Affinity-purified antibodies against Xorc2, Xchk1, and Claspin were described previously (Coleman et al., 1996; Kumagai et al., 1998; Kumagai and Dunphy, 2000). Antibodies against Xenopus RPA70, Xatr, and Xrad17 were raised against His6-tagged fragments containing amino acids 109–410, 1–480, and 443–674 of these proteins, respectively. For anti-Xhus1 antibodies, the whole Xhus1 protein was made in a His6-tagged form. Anti-RPA70, anti-Xatr, anti-Xrad17, and anti-Xhus1 antibodies were affinity purified with their antigens. Antisera against Xcdc45 and Xmcm7 were generously supplied by J. Walter and J. Blow, respectively (Prokhorova and Blow, 2000; Walter and Newport, 2000). Antisera that detect p70 of Pol ␣ and p60 of Pol ⑀, respectively, were kindly provided by S. Waga (Waga

et al., 2001). Antisera against the p36, p37, p38, and p40 subunits of human RFC were the kind gift of J. Hurwitz (Uhlmann et al., 1996). Anti-human Chk1 phospho-Ser345 antibodies, which recognize the Ser344-phosphorylated form of Xchk1, were purchased from Cell Signaling Technology. Anti-human PCNA antibodies and purified control rabbit IgG were obtained from Pharmingen and Zymed, respectively. Immunodepletions Immunodepletions of Claspin, Xcdc45, and RPA70 from egg extracts were carried out with Affiprep-protein A beads (Bio-Rad) as described previously (Kumagai and Dunphy, 2000; Walter and Newport, 2000). Xatr and Xrad17 were removed from extracts with antibody-coated protein A-magnetic beads (Dynal). The magnetic beads were incubated in extracts for 1 hr on ice. The beads were removed with a magnet and the procedure was repeated. Isolation of Nuclear and Chromatin Fractions For isolation of nuclear fractions, egg extracts (50 ␮l) containing 3000 sperm nuclei/␮l were incubated, overlaid on a 1 ml sucrose cushion (20 mM HEPES-KOH [pH 7.6], 1 M sucrose, 80 mM KCl, 2.5 mM K-gluconate, and 10 mM Mg-gluconate), and centrifuged at 6100 g for 5 min. The pellets were resuspended and centrifuged again. The nuclear fractions were dissolved in gel sample buffer. For preparation of chromatin fractions, sperm nuclei (3000/␮l) were incubated for 90 min in extracts. Next, 50 ␮l aliquots of the extracts were removed, chilled on ice, and diluted in 0.5 ml of ice-cold chromatin isolation buffer (20 mM HEPES-KOH [pH 7.6], 80 mM KCl, 2.5 mM K-gluconate, 10 mM Mg-gluconate, 0.5% NP-40, and 1 mM DTT). The diluted extracts were overlaid on a 0.5 ml cushion containing sucrose (1 M) in chromatin isolation buffer and centrifuged at 6100 g for 5 min. The entire supernatant was removed and the chromatin pellet was subjected to SDS-PAGE. Binding of Proteins to Immobilized Oligonucleotides A 100 ␮l suspension of Streptavidin-conjugated magnetic beads (Dynal) was incubated with 10 ␮g of the biotinylated poly(dA)70 in a final volume of 200 ␮l of binding buffer (10 mM Tris-HCl [pH 7.6], 1 M NaCl, and 1 mM EDTA). After 30 min at 23⬚C, the beads were washed with TE (10 mM Tris-HCl [pH 7.6] and 1 mM EDTA) and resuspended in 40 ␮l of TE. Aliquots (5 ␮l) of immobilized poly(dA)70 on the beads were incubated in 100 ␮l of egg extract for 90 min at 23⬚C. The beads were collected by centrifugation through a sucrose cushion. The pellets were washed twice with ice-cold buffer (10 mM HEPES-KOH [pH 7.6], 80 mM NaCl, 0.1% NP-40, 2.5 mM EGTA, and 20 mM ␤-glycerolphosphate) and collected each time with a magnet. Acknowledgments We thank our laboratory for helpful comments. We would like to thank J. Blow, A. Dutta, J. Hurwitz, T. McGarry, H. Takisawa, S. Waga, J. Walter, and M. Wold for kindly providing the valuable reagents described in the text. We are grateful to C. Pham and Y. Li for their contributions. These studies were supported in part by a grant from the NIH (GM43974). J.L. is an associate and W.G.D. is an investigator in the Howard Hughes Medical Institute. Received: September 17, 2002 Revised: November 14, 2002 References Abraham, R.T. (2001). Cell cycle checkpoint signaling through the ATM and ATR kinases. Genes Dev. 15, 2177–2196. Alcasabas, A.A., Osborn, A.J., Bachant, J., Hu, F., Werler, P.J., Bousset, K., Furuya, K., Diffley, J.F., Carr, A.M., and Elledge, S.J. (2001). Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 3, 958–965. Bao, S., Tibbetts, R.S., Brumbaugh, K.M., Fang, Y., Richardson, D.A., Ali, A., Chen, S.M., Abraham, R.T., and Wang, X.F. (2001). ATR/ATM-mediated phosphorylation of human Rad17 is required for genotoxic stress responses. Nature 411, 969–974.

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