| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
1 Department of Bioscience, Graduate School of Science, Osaka University, Toyonaka, Osaka 560-0043, Japan
2 Laboratories for Biomolecular Networks, Graduate School of Frontier Biosciences, Osaka University, Suita, Osaka 565-0871
 |
Abstract |
|---|
 Top Abstract IntroductionResultsDiscussionExperimental proceduresReferences |
|---|
|
Introduction |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
To activate the replication checkpoint, the initiation of DNA replication is essential, and the initiation is a highly regulated process through the progression of the cell cycle. During the late M and G1 phases, ORC, Cdc6, Cdt1, and MCM are sequentially assembled on to replication origins. This leads to the formation of the prereplicative complex (pre-RC), eventually establishing origin licensing (Blow & Hodgson 2002; Nishitani & Lygerou 2002). At the onset of the S-phase, the pre-RC is converted into the preinitiation complex (pre-IC) through the action of two protein kinases, CDK and DDK. CDK also plays an important role in preventing re-licensing until exit from the M-phase (Diffley 2004). Emerging evidence suggests that various factors are required for the formation of the pre-IC, including Cut5/Dpb11, Sld2/Drc1, Sld3, GINS, and Cdc45 (Kearsey & Cotterill 2003; Méndez & Stillman 2003). After the formation of the pre-IC, the unwinding of origin DNA leads to sequential recruitments of replication protein A (RPA), DNA polymerase 
(Pol), replication factor C (RFC), proliferating cell nuclear antigen (PCNA) and Pol
(Mimura et al. 2000; Walter & Newport 2000). Pol
is also recruited to the origin, but independently of RPA (Mimura et al. 2000). The replication checkpoint system monitors the replication forks, where Pol synthesizes RNA-DNA primers and Pol and Pol, respectively, synthesize the leading and lagging strands of DNA.
When the progression of the replication forks is perturbed, the upstream kinase ATR is activated to phosphorylate the downstream kinase Chk1, leading to its activation. The activated Chk1 then phosphorylates its substrate to inhibit mitotic entry (O’Connell & Cimprich 2005). The formation of excessively long ssDNA, which is normally coated with RPA, is thought to be critical for the activation of ATR (Zou & Elledge 2003). The region of ssDNA greatly increases when DNA replication is inhibited by DNA replication inhibitors such as aphidicolin and HU, or by DNA damage inducers such as methyl methanesulfonate or UV light (Sogo et al. 2002; Zou & Elledge 2003; Byun et al. 2005). Such treatments are believed to cause an uncoupling of the activities between replicative polymerases and helicase, resulting in the accumulation of ssDNA (Byun et al. 2005). ATR associates in vitro with RPA-ssDNA complexes through its partner protein ATRIP (Zou & Elledge 2003; Kim et al. 2005a). Checkpoint proteins such as claspin/Mrc1, the Rad17/RFC complex, and the Rad9/Rad1/Hus1 (9-1-1) complex are also required for ATR-dependent activation of Chk1 (O’Connell & Cimprich 2005). Rad17/RFC loads the 9-1-1 complex on to primed DNA in vitro (O’Connell & Cimprich 2005). Although ATR and Rad17 are both dependent on RPA for their chromatin binding, these two proteins bind to chromatin independently of each other (You et al. 2002; Zou et al. 2002; Lee et al. 2003), suggesting that they recognize different structures at the stalled replication fork. Claspin/Mrc1 constitutively associates with replication forks, forming a stable replication-pausing complex together with Tof1 and Csm3 (Katou et al. 2003; Osborn & Elledge 2003; Calzada et al. 2005). In addition, phosphorylated claspin interacts with Chk1 and mediates its activation (Jeong et al. 2003; Kumagai & Dunphy 2003; Kumagai et al. 2004). Thus, the concerted interaction of multiple proteins constitutes a monitoring system for DNA replication.
Cut5/Dpb11/TopBP1 is a multifunctional protein, implicated in many aspects of DNA metabolism, including DNA replication initiation, DNA damage and replication checkpoints, and transcriptional regulation (Garcia et al. 2005). The role in initiation has been extensively studied in yeasts, clarifying that Cut5/Dpb11 associates with Sld2/Drc1 phosphorylated by CDK, and the complex is key to the formation of pre-IC (Masumoto et al. 2002; Noguchi et al. 2002). As with yeast Dpb11/Cut5, Xenopus Cut5 is required for the action of CDK in replication initiation (Hashimoto & Takisawa 2003). In DNA damage response, Schizosaccharomyces pombe Cut5 and human TopBP1 are required by the G2/M damage checkpoint, where Cut5/TopBP1 collaborates with phosphorylated Rad9 to activate Chk1 (St Onge et al. 2003; Furuya et al. 2004). A conserved interaction of Saccharomyces cerevisiae Dpb11 with Rad9/Ddc1 (Wang & Elledge 2002) further suggests the conserved function of Cut5 homologs in damage response. Upon DNA damage, human TopBP1 is phosphorylated at several S/TQ sites, which are consensus sequences of PIKK targets, but the role of the phosphorylation is not understood (Yamane et al. 2002). In contrast to its role in the damage checkpoint, it is difficult to investigate the role of Cut5 homologs in the replication checkpoint because they are essential for the formation of replication forks (Wang & Elledge 2002; Mochida et al. 2004). In fact, no checkpoint-specific mutations in Cut5/Dpb11 have been reported. Previous studies clearly show that Cut5/Dpb11/TopBP1 is involved in the replication checkpoint (Saka et al. 1994; Araki et al. 1995; Wang & Elledge 1999; Parrilla-Castellar & Karnitz 2003; Kim et al. 2005b), but its exact function at stalled replication forks is largely unknown. Furthermore, metazoan Cut5/TopBP1 has a different molecular structure than yeast Cut5/Dpb11 (Garcia et al. 2005). Namely, metazoan Cut5/TopBP1 consists of two regions: the N-terminal half region is conserved through evolution, while the C-terminal half region is unique in metazoans. It has not been clarified whether the whole region is required for replication or the checkpoint function.
In this study, we aimed to identify the role of Cut5 in the replication checkpoint using Xenopus egg extracts. By dissecting the functional region, we identified that the conserved N-terminal half region is required for the initiation reaction, and that the unique C-terminal half region is required for the checkpoint reaction. We further found that recombinant Cut5 stimulated ATR-dependent phosphorylation of Chk1 in vitro, and that its ability to mediate ATR-Chk1 signaling was regulated by the phosphorylation of Cut5 on Ser1131. These results are the first demonstration that Cut5 phosphorylation directly mediates ATR-Chk1 signal transduction in the replication checkpoint.
|
Results |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
The N-terminal halves of Xenopus Cut5, Drosophila Mus101, and human TopBP1 show good sequence similarity to the full-length yeast Cut5/Dpb11, but the C-terminal half-domains are unique to the higher eukaryotes (Fig. 1A; see also Hashimoto & Takisawa 2003). To explore the functions of the yeast-homologous domain of Xenopus Cut5 and the unique domain, we produced N- and C-terminal halves of Xenopus Cut5 as recombinant proteins of similar lengths. We then examined the function of these peptides with interphase egg extracts. When endogenous Cut5 was depleted, the replication activity of sperm nuclei added to the extracts was almost abolished (Fig. 1B,D), and the chromatin binding of various replication proteins, except Mcm3, was suppressed (Fig. 1C), as reported previously (Van Hatten et al. 2002; Hashimoto & Takisawa 2003). The replication activity of the depleted extracts could be rescued by the full-length protein (Cut5-F), as previously described; and, intriguingly, the N-terminal half of Cut5 (Cut5-N), but not the C-terminal half (Cut5-C), could also efficiently rescue the replication activity (Fig. 1B,D). In parallel with the replication activity, Cut5-N and Cut5-F but not Cut5-C were able to restore the chromatin binding of the replication-related proteins Cdc45, Pol, Pol, and claspin (Fig. 1C). The binding of endogenous Cut5 as well as other replication proteins was apparently unaffected by further addition of Cut5-N or Cut5-C to the mock-depleted extracts. These results indicate that the N-terminal half-domain of Cut5 is sufficient for DNA replication.
|
|
The remaining question is whether Cut5-C is sufficient for the phosphorylation of Chk1 at replication block. To circumvent the inability of Cut5-C to initiate DNA replication, we used chromatin transfer experiments, which allowed us to examine the replication checkpoint in the absence of the initiation reaction. The replicating chromatin fractions were isolated with high-salt buffer to remove chromatin-bound Cut5 and then transferred to Cut5-depleted extract supplemented with aphidicolin to block elongation activity and with CDK inhibitors (p21 and roscovitine) to inhibit firing of new origins, thus keeping the fork number constant. Salt treatment affected neither the chromatin binding of replication proteins such as MCM, Cdc45, DNA polymerases, and RPA (Supplementary Fig. S1B), nor the replication activity of established replication forks (see Fig. 4 in Hashimoto & Takisawa 2003), but nearly all Cut5 was removed from the chromatin (Supplementary Fig. S1B,C,E). As shown in Fig. 2C, aphidicolin-induced Chk1 phosphorylation was detected when the washed replicating chromatin was transferred to mock-depleted extracts, but it was hardly detectable when the chromatin was transferred to Cut5-depleted extracts. The phosphorylation of Chk1 in Cut5-depleted extracts could be rescued by Cut5-F and, more importantly, by Cut5-C, which rescued the Chk1 phosphorylation at a similar level to that with Cut5-F. These results strongly suggest that the C-terminal half of Cut5 is sufficient for the ATR-dependent activation of Chk1 at replication block. It should also be noted that most of the ATR and claspin was removed from salt-treated chromatin (Supplementary Fig. S1B), but they could re-associate with chromatin in the second incubation in the absence of Cut5 (Supplementary Fig. S1E). These data indicate that Cut5 is not required for the chromatin binding of ATR and claspin at replication block, but it is required for the signal transduction between ATR and Chk1.
|
Previously we reported that Xenopus Cut5 is bound to chromatin at a low level before the initiation of DNA replication (CDK-independent binding), and the binding greatly increases after the initiation (CDK-dependent binding) (Supplementary Fig. S2; see also Hashimoto & Takisawa 2003). CDK-independent binding is sufficient for the initiation of DNA replication, but CDK-dependent binding is apparently dispensable (Fig. 3B; and see also Hashimoto & Takisawa 2003). Since most Cut5 is bound to chromatin after the initiation reaction that depends on CDK activity, we speculate that the CDK-dependent binding is involved in the checkpoint function. To restrict the Cut5 binding to CDK-independent mode, we incubated sperm nuclei in interphase egg extracts in the presence of CDK inhibitors for 15 min and then transferred the isolated chromatin fractions to the mock- or Cut5-depleted egg extracts and examined the response to replication block (Fig. 3A). As previously reported, the replication activity of the transferred chromatin was essentially the same in mock- and Cut5-depleted extracts (Fig. 3B), but the caffeine-sensitive phosphorylation of Chk1 in response to aphidicolin block was largely compromised in the Cut5-depleted extracts (without CDK-dependent binding, and thus only a small amount of Cut5 remained on chromatin in CDK-independent binding) (Fig. 3C). The immunoblotting of the chromatin fractions showed that only a small amount of Cut5 was bound to chromatin in Cut5-depleted extracts, and both the mock- and Cut5-depleted extracts supported the chromatin binding of the replication and checkpoint proteins Orc1, Mcm3, RPA70, Cdc45, Pol and Pol, ATR, and claspin at similar levels in both the presence and absence of aphidicolin (Fig. 3D). These results suggest that CDK-dependent binding is not required for the chromatin binding of those factors, and that the replication machinery apparently remained stable regardless of the CDK-dependent binding. Taken together, these results suggest that the CDK-dependent binding is engaged in the checkpoint function under physiological conditions.
|
CDK-dependent binding of Cut5 is probably required for full activation of the replication checkpoint, but it is not required for the chromatin binding of various replication and checkpoint proteins, including RPA and ATR (Fig. 3), both of which are crucial components of the checkpoint response. Thus, we examined whether the binding of these proteins occurs upstream of the CDK-dependent binding of Cut5 (Fig. 4). To exclude the possible involvement of DNA damage upon replication block, we measured the binding after 40-min incubation of sperm chromatin in egg extracts without addition of aphidicolin, when CDK-dependent binding was near maximum. With mock-depleted extracts, CDK-dependent binding was apparent, and replication proteins Pol, Pol, and Cdc45 were loaded on to chromatin (Fig. 4A). With Cut5-depleted extracts, most replication proteins were diminished on the chromatin except for Mcm3 and RPA. Similar amounts of Mcm3, Pol, and Cdc45 were loaded on to chromatin in both mock- and RPA-depleted extracts, in accord with our previous report (Mimura et al. 2000). Depletion of RPA severely suppressed the chromatin binding of Pol and, more importantly, that of Cut5. However, RPA is not apparently required for the CDK-independent binding of Cut5 implicated in the formation of pre-IC, because both Cdc45 and Pol, components of pre-IC, are loaded on to chromatin in RPA-depleted extracts. Therefore, these results suggest that RPA is required for the CDK-dependent binding of Cut5 implicated in the checkpoint.
In checkpoint reactions, RPA is essential for the chromatin association of ATR, which is thought to be a crucial step in the propagation of the signaling cascade (You et al. 2002; Lee et al. 2003). We therefore examined whether ATR is required for Cut5 binding (Fig. 4B). After depleting ATR from the extracts, we detected no chromatin binding of ATR in the presence or absence of aphidicolin, but the chromatin binding of Cut5 was virtually unaffected when compared with that in the mock-depleted extracts. Similarly, the chromatin binding of replication proteins RPA, Cdc45 and Pol was not affected by ATR depletion. The chromatin binding of Cut5 and RPA was similarly increased upon replication block in both the presence and absence of ATR. These results show that RPA, but not ATR, is closely involved in the CDK-dependent binding of Cut5.
Phosphorylation of the C-terminal domain of Cut5 in the checkpoint response
ATR/ATM-dependent phosphorylation of checkpoint proteins plays an important role in controlling the signaling pathway. Vertebrate Cut5/TopBP1 has several S/TQ motifs, putative target sites of PI3-kinases (Kim et al. 1999), and several S/TQ sites of human TopBP1, including S405Q, S467Q, and S1051Q, are phosphorylated by ATM, but their physiological role remains unknown (Yamane et al. 2002). Two of the three sites are conserved in Xenopus Cut5; they correspond to S488Q and S1131Q (Fig. 5A). We examined whether S1131Q, located in the C-terminal checkpoint domain, is phosphorylated during checkpoint activation by producing phospho-specific antibody against a synthetic phosphorylated peptide of 16 amino acids around Ser1131 (a.a. 1127–1142). To detect the phosphorylation, we immunoprecipitated Cut5 with antibody against full-length Cut5 protein from egg extracts or nuclear extracts (Fig. 5B). The total amounts of Cut5, which is detected by normal antibody, were comparable in all of the immunoprecipitates. The phospho-specific antibody recognized Cut5 prepared from the extracts only when the checkpoint response was activated: in the egg extracts containing the annealed oligonucleotides and in those prepared from aphidicolin-treated nuclei (lanes 3 and 7). These signals disappeared when the precipitates were treated with phosphatase in the absence of the inhibitor (lanes 4 and 8), showing that the signals were indeed derived from the phosphorylation of Cut5. Caffeine applied with wortmannin, which inhibits three PIKK members (ATR, ATM, DNA-PK), also diminished the signal in the nuclear extracts (lane 9). If we assume the specificity of these inhibitors to PIKK kinases, these results suggest that PIKK family kinases phosphorylate Cut5-Ser1131 when the checkpoint response has been activated.
|
Essentially the same role of Ser1131 modification in the checkpoint response was observed at replication block in the presence of sperm nuclei (Fig. 5D). Either Cut5-WT or Cut5-1131E was able to rescue the phosphorylation of Chk1 upon replication block, while Cut5-1131 A could only poorly rescue the activity in endogenous Cut5-depleted extracts. Cut5-1131 A also inhibited Chk1 phosphorylation in the mock-depleted extracts, indicating that the mutant protein has a dominant negative form. The phenotype of the mutant proteins was not due to the defect of replication initiation, because Cut5-1131 A supported the replication activity and the formation of the replication complex at comparable levels as did Cut5-WT and Cut5-1131E (Supplementary Fig. S3). These results therefore demonstrate that the phosphorylation of Cut5 on Ser1131 plays a crucial role in the checkpoint response at replication block.
Direct activation of ATR-Chk1 pathway by Cut5
The essential requirement of Ser1131 phosphorylation for the activation of Chk1 raises the possibility that Cut5 is directly involved in the control of ATR activity. To examine this possibility, we examined the effect of recombinant Cut5 on the phosphorylation of Chk1-Ser344 in vitro by immuno-purified ATR from egg extracts. We used as a substrate a kinase-dead form of recombinant Chk1 (Chk1-KD), whose activity was inactivated by a point mutation to suppress autophosphorylation (Shimuta et al. 2002). A very low level of phosphorylation of the Ser344 of Chk1-KD was detected when Chk1-KD was incubated with immunoprecipitates from the egg extracts using control antibody (data not shown and see also Fig. 6A, lanes 1 & 2), and a low but significant level of phosphorylation was detected by incubating Chk1-KD with the affinity-purified ATR from the extracts (Fig. 6A
-ATR-IP, lane 3, and Fig. 6C, lane 1). Further addition of Cut5-WT or Cut5-1131E to the affinity-purified ATR stimulated the phosphorylation of Chk1-KD in a dose-dependent manner (Fig. 6A, lanes 4-6 and 10-12). But the addition of Cut5-1131 A hardly stimulated the phosphorylation of Chk1, and the level of phosphorylation remained similar to that observed in the absence of Cut5 (compare Fig. 6A lane 3 and lanes 7-9). Neither the Cut5-WT nor Cut51131E alone stimulated the phosphorylation of Chk1-KD, as clearly depicted by the very low level of phosphorylation of Chk1-KD with the control immunoprecipitates (Fig. 6A, lanes 1 & 2). When we take the phosphorylation level detected in the control precipitates as background, the obtained data indicate that the phosphorylation of Chk1 in vitro is actually activated by Cut5, and the phosphorylation of Cut5-Ser1131 is essential for the phosphorylation of Chk1.
|
|
Discussion |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Functional domain of Xenopus Cut5/TopBP1
We successfully separated the checkpoint function from the initiation function of Cut5 by dissecting the protein, and found that the distinct modules are independently engaged in each function (Fig. 7). The N-terminal half of Xenopus Cut5, which shows good sequence similarity to the full-length yeast Cut5/Dpb11, is responsible for the initiation function, but failed to activate the checkpoint response (Figs 1 and 2). The intrinsic ability of the N-terminal half region to initiate replication clearly demonstrates that Xenopus Cut5 is the authentic homolog of yeast Cut5/Dpb11 in DNA replication. In contrast, the C-terminal half is dispensable for the replication, and its absence apparently stimulated replication (Fig. 1). We found that the C-terminal half is sufficient for ATR-dependent Chk1 phosphorylation induced by either replication block or synthetic oligonucleotides (Fig. 2), showing that the C-terminal region is responsible for the checkpoint reaction. Since the ATR/ATM pathway regulates the timing of DNA replication origin firing (Marheineke & Hyrien 2004; Shechter et al. 2004), the apparent activation of DNA replication in the absence of the C-terminal region suggests that this region is involved in the control of DNA replication timing in addition to the replication checkpoint. The unique C-terminal half of human TopBP1 interacts with topoisomerase IIß, Miz-1, and E2F-1 (Garcia et al. 2005). Thus, vertebrate Cut5/Mus101/TopBP1 might have different functions from those of yeast homologs. It is also possible to speculate that additional factors in yeasts play a role similar to that of the C-terminus of Xenopus Cut5. In this regard, it is tempting to speculate that the C-terminal domain functions like S. pombe Crb2/Rhq9, which contains tandem BRCTdomains and interacts with Cut5 in the DNA damage response (Saka et al. 1997; Mochida et al. 2004), although the importance of Crb2 in replication stress has not been established.
|
Previous studies show that RPA is required for the chromatin association of ATR (You et al. 2002; Lee et al. 2003), and that ATR-ATRIP directly recognizes the RPA-ssDNA complex in vitro (Zou & Elledge 2003; Kim et al. 2005a). We found that the CDK-dependent chromatin binding of Cut5, which is possibly implicated in the checkpoint response, requires RPA but not ATR in egg extracts (Fig. 4), and that Cut5 is not required for the re-association of ATR to chromatin after high-salt wash (Fig. S1). These results suggest that Cut5 and ATR independently bind to chromatin in the presence of RPA. A previous study using etoposide (Parrilla-Castellar & Karnitz 2003) reported the sequential binding of RPA, Cut5, and ATR to chromatin in extracts. The different requirement of Cut5 for ATR binding might be due to the distinct action of etoposide from that of aphidicolin. Claspin, on the other hand, binds to chromatin independently of ATR and RPA, but the binding is dependent on Cdc45 (Lee et al. 2003). Therefore, we suggest the following order of chromatin binding of various replication/checkpoint proteins: (i) CDK-independent mode of Cut5, (ii) Cdc45, (iii) claspin and RPA, (iv) ATR and CDK-dependent mode of Cut5. We previously reported that Cut5 binding gradually increases even in the absence of Cdc45 (Hashimoto & Takisawa 2003), when RPA binding to ssDNA is suppressed because of the lack of DNA unwinding (Pacek & Walter 2004). The exact mechanism of Cdc45-independent Cut5 binding is not yet known, but such increased binding without DNA replication may not be related to physiological intermediates. In contrast, the RPA-dependent binding of Cut5 found in the present study suggests that Cut5 recognizes DNA structures coated with RPA arising from perturbed and unperturbed replication forks.
Two modes of Cut5 binding found with the extracts in the embryonic cell cycle may play a similar role in the somatic cell cycle. The amount of CDK-independent binding implicated in the initiation is relatively small, compared with the amount of CDK-dependent binding implicated in the checkpoint (Fig. 3; and see also Hashimoto & Takisawa 2003). Thus, only a small amount of Cut5 is required for initiating DNA replication, and most of Cut5 is required for the checkpoint response. In accord with this, the phenotype of TopBP1 knock-down cells created by small interfering RNA (Kim et al. 2005b) shows no defect in replication initiation, but instead shows the accumulation of DSBs possibly created during DNA replication. Therefore, the majority of cellular Cut5/TopBP1 is most likely engaged in the checkpoint function during DNA replication. This suggests that Cut5 binds to chromatin in the two modes in somatic cells. However, we could not exclude the possibility that the amount of Cut5 required for the checkpoint is much higher than that for the initiation. Further study will clarify the physiological role of the two modes of chromatin binding of Cut5 in egg extracts as well as in somatic cells.
Role of Cut5 in the replication checkpoint
The ATR-Chk1 signal transduction pathway plays a central role in the replication checkpoint. Thus far, many factors have been suggested to function as sensors or mediators to help the activation of the pathway. Among them, Cut5, claspin, Rad17/RFC, and Rad9/Hus1/Rad1 (9-1-1) are conserved throughout eukaryotic evolution. In this study, we have demonstrated that Cut5 has a stimulatory effect on ATR-dependent phosphorylation of Chk1-KD with an in vitro assay using recombinant proteins and immunopurified ATR (Fig. 6). We also found that claspin alone scarcely facilitated the same reaction, but the simultaneous addition of Cut5 and claspin further enhanced the phosphorylation of Chk1, compared with Cut5 alone. According to Kumagai et al. (2004), Chk1 phosphorylation occurs at a weak level in the absence of claspin. More significantly, claspin can induce autophosphorylation of Chk1 even in the absence of ATR-ATRIP (Kumagai et al. 2004). Taken together, these results support a model in which Cut5 mediates the primary step of Chk1 activation by ATR, and claspin mediates the autophosphorylation of primed Chk1, converting it to fully active Chk1 as an effector kinase (Fig. 7).
Rad17/RFC and 9-1-1 complex are also required for the checkpoint activation. Rad17/RFC recognizes primed DNA structures and loads 9-1-1 complex in vitro (O’Connell & Cimprich 2005), but there is no evidence that they are directly involved in the phosphorylation of Chk1. Previous studies suggest that Rad9 interacts with Cut5/TopBP1, depending on the phosphorylation of the Rad9 C-terminus (St Onge et al. 2003; Furuya et al. 2004). Therefore, Cut5/TopBP1 associates with Rad9, which recognizes aberrant DNA structures at a stalled replication fork, and then mediates the activation of the ATR-Chk1 pathway together with another fork protein, claspin. In this regard, Rad17 and Rad9/Hus1/Rad1 behave differently from Cut5 and claspin in chromatin binding. Cut5 and claspin bind to unperturbed replicating chromatin and aphidicolin-treated chromatin at a similar level (Fig. 3D and Supplementary Fig. S2) (Lee et al. 2003). In contrast, Rad17 and 9-1-1 are scarcely detected on unperturbed replicating chromatin, and the chromatin binding markedly increases at replication block (Lupardus et al. 2002; You et al. 2002; Jones et al. 2003; Lee et al. 2003). These results suggest that Cut5 and claspin constitutively monitor the progression of the replication fork, while Rad17 and 9-1-1 are mobilized specifically in response to a stalled fork. Although the role of the interaction between Cut5 and 9-1-1 is still unclear, it might maintain the checkpoint signaling complex in an active state to fully activate the replication checkpoint.
Regulation of the checkpoint function of Cut5 through phosphorylation
We found that the phosphorylation of the SQ site of Cut5 is involved in the regulation of its activator function (Figs 5 and 6). Xenopus Cut5 has eight BRCT domains, which are recognized as phospho-S/TQ peptide-binding motifs (Manke et al. 2003; Yu et al. 2003). This raises an interesting possibility that the phosphorylated S1131Q in Cut5 is recognized by its own BRCT domains to induce inter or intramolecular structural changes. At present we could not detect any conformational changes in mutant Cut5 (S1131E) or any difference in the molecular assembly between wild and mutant proteins by glycerol gradient sedimentation experiments (data not shown). An alternative possibility is that the phosphorylation site is targeted by other binding partners such as Rad9 (Garcia et al. 2005). However, our in vitro kinase assay shows that non-phosphorylatable (1131 A) and phospho-mimetic (1131E) mutants of Cut5 have negative and positive effects consisting of the anti-ATR-immunoprecipitates, recombinant Cut5 and Chk1-KD. Thus, the simplest explanation for the positive effect of the phosphorylation at Ser1131 is that the phosphorylation affects the interaction between ATR/ATRIP, Chk1, and Cut5.
A remaining question is what kind of kinase is responsible for the phosphorylation of Cut5 on replication block. S488Q and S1131Q in Xenopus Cut5 are conserved in human TopBP1 (S405Q and S1051Q), which were originally identified as ATM-dependent phosphorylation sites in vitro (Yamane et al. 2002). Since the S/TQ sequences are recognized as a consensus target motif for three PIKK kinases (ATR, ATM, DNA-PK) (Kim et al. 1999), it is likely that each of them could phosphorylate Cut5 at Ser1131. In fact, we detected the phosphorylation signal at Ser1131 of Cut5 in the presence of either caffeine, an ATR/ATM inhibitor, or of wortmannin, an ATM/DNA-PK inhibitor, and even with the ATR-depleted extracts (data not shown). The phosphorylation signal was abolished in the presence of both caffeine and wortmannin (Fig. 5), suggesting that the three PIKK kinases can phosphorylate the Ser1131 of Cut5. However, it is not known yet whether only the phosphorylation of Ser1131 of Cut5 is involved in the regulation of its activity, because the phosphorylation of Chk1 is completely abolished by the addition of caffeine to the extracts. The identity of the kinase(s) responsible for the phosphorylation of Cut5 to regulate its activity remains to be determined.
While we were preparing this manuscript, a paper was published showing that human and Xenopus TopBP1/Cut5 directly activate ATR (Kumagai et al. 2006). Kumagai et al. (2006) clearly show that TopBP1 is involved in the ATR activation, which is a similar finding to ours. In addition, they identified the ATR-activating domain as being in the C-terminal half of TopBP1. We found that the initiation function of Cut5/TopBP1 could be clearly separated from its checkpoint function, and the phosphorylation of serine 1131 within the putative activating domain is essential for its checkpoint activity. Since serine 1131 is phosphorylated during replication block in vivo, the phosphorylation of Cut5/TopBP1 should be a crucial step in regulating the checkpoint function of Cut5/TopBP1 in higher eukaryotes. In addition, it remains to be determined whether or not Cut5/TopBP1 acts solely as an activator of ATR or also acts as mediator, which has been suggested with yeast Cut5 (Garcia et al. 2005).
Note
During the revision of our manuscript, Yan et al. (2006) reported that Xenopus Mus101/Cut5/TopBP1 is directly required in ATR-mediated phosphorylation of Chk1 during checkpoint signaling.
|
Experimental procedures |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
Affinity-purified antibodies against Cut5, Cdc45, Pol, RPA70, Mcm3, and Orc1 were prepared as previously described (Mimura & Takisawa 1998; Mimura et al. 2000; Hashimoto & Takisawa 2003). For anti-ATR antibody, antisera against an N-terminal peptide of Xenopus ATR (MATDPGLEMASMIPALRELC, residues 1-20) (Hekmat-Nejad et al. 2000) and an internal peptide (EKTNPKPGTRGEPK, residues 1617-1630) (Guo et al. 2000) were generated (Sawady Technology, Japan) and further affinity-purified with the peptide antigens. For anti-Chk1 and anti-Pol (p60) antibodies, antisera were raised against GST-tagged forms of the full-length Xenopus Chk1 and the full-length Xenopus Pol
(p60) (the expression vector was a generous gift from Dr Waga (Osaka University, Osaka, Japan; Waga et al. 2001) and further affinity-purified with those antigens. For anti-claspin antibody, antiserum was raised against the His-tagged form of the full-length claspin (the cDNA was a generous gift from Dr Dunphy (California Institute of Technology, Pasadena, CA, USA; Kumagai & Dunphy 2000) and further affinity-purified with the antigen. To detect the phosphorylated form of Xenopus Chk1, we purchased an anti-human Chk1 phospho-Ser345 antibody that reacts with the Ser344-phosphorylated form of Xenopus Chk1 (Cell Signaling Technology). For the antibody to detect the phosphorylated form of Xenopus Cut5, antiserum against a phosphorylated peptide (NTEP(phospho-S)QNEIIWDDPT, residues 1127-1142) was affinity-purified with the antigen and further absorbed with an unphosphorylated version of the same peptide.
Xenopus egg extracts and nuclear fraction
S-phase egg extracts and de-membranated sperm nuclei were prepared as described (Kubota & Takisawa 1993). Aphidicolin was added to the egg extracts at a concentration of 20–40 µg/mL to inhibit DNA polymerases. GST-p21 (Mimura & Takisawa 1998) and roscovitine (Sigma) were added to the extracts at 50 µg/mL and 100 µM, respectively, to inhibit S-CDK. GST-geminin (Mimura et al. 2000) was used at 15 µg/mL to inhibit the licensing reaction. Caffeine was added at 5 mM to inhibit the kinase activities of ATR/ATM. Wortmannin (Sigma) was added at 100 µM to inhibit the kinase activities of ATM/DNA-PK. The nuclear fractions were prepared as described (Kumagai et al. 1998) to detect the phosphorylated form of endogenous Chk1.
Chromatin isolation and chromatin transfer
To isolate the chromatin fractions, we incubated sperm nuclei in 25-50 µL of the egg extracts (5000 nuclei/µL extract) for appropriate times as described in the figure legends. The samples were treated as follows: For immunoblotting, samples were diluted with 10-20 volumes of EB (100 mM KCl, 2.5 mM MgCl2, and 50 mM HEPES-KOH, pH 7.5) containing 0.25% NP40, and then centrifuged through a 20% sucrose layer at 10 000 g at 4 °C for 5 min; the pellets were washed with EB. The pellets were suspended in SDS-PAGE sample buffer, and the suspensions were filtered through a 0.45-µm nitrocellulose filter to remove DNA. For the chromatin transfer experiments, the samples obtained after 15 min preincubation were diluted with 10–20 volumes of EB containing 2 mM dithiothreitol, and then centrifuged through a 20% sucrose layer at 10 000 g at 4 °C for 5 min; the pellets were washed with EB and re-suspended in egg extracts. The samples obtained after 45 min preincubation were treated as described above except that 0.0025% NP40 was included in the dilution buffer to permeabilize the nuclear membrane.
Immunodepletion, immunoprecipitation, and immunofluorescence
Immunodepletion, immunoprecipitation, and immunofluorescence were carried out as described (Hashimoto & Takisawa 2003). For the immunodepletion from 100 µL of Xenopus egg extracts, 60 µg of anti-Cut5 antibody, 140 µg of anti-ATR antibody (anti-internal peptide), or 140 µg of anti-RPA antibody was used. For phosphatase treatment, the immunoprecipitated beads from 20 µL of egg extracts were washed with EB and incubated with 5 units of calf intestine alkaline phosphatase (Roche) in 20 µL of dephosphorylation buffer (pH 8.5) containing protease inhibitors (10 µg/mL each of leupeptin, aprotinin, anti-pain, and pepstatin) in the presence or absence of 20 mM sodium phosphate at 30 °C for 1 h. After washing with EB, proteins bound to the beads were immunoblotted.
Protein expression
The recombinant protein of full-length Xenopus Cut5 was prepared as described (Hashimoto & Takisawa 2003). For the production of the N-terminal half region of Xenopus Cut5, the cDNA fragment coding the C-terminal region (corresponding to amino acids 759-1513) spanning the XbaI and XhoI sites was removed from the pFastBac HTb plasmid carrying the full-length ORF of Xenopus Cut5, and then the digested plasmid sites were blunted with T4 DNA polymerase and self-ligated. Similarly, for the production of the C-terminal region, the cDNA fragment coding the N-terminal region (amino acid 2-757) spanning the NcoI and XhaI sites was removed from the full-length plasmid, and the digested sites were blunted and self-ligated. These plasmids were used to produce the recombinant proteins of the N-terminal and C-terminal half regions of Xenopus Cut5. To create point mutants of Xenopus Cut5, we mutated the HTb plasmids carrying the full-length Cut5 cDNA by PCR so that the codon for Ser1131 was changed to that for Ala or Glu, respectively. The cDNA encoding a mutant Chk1 (kinase-dead form of Chk1: Chk1-KD) in which Asp148 was replaced with Ala was a generous gift from Dr Sagata (Kyushu University, Fukuoka, Japan; Shimuta et al. 2002), and the His-tagged version was produced by subcloning the cDNA into pFastBac HTb.
Kinase assay
Two micrograms of each anti-ATR antibody was conjugated with 5 µL of Affi-Prep Protein A matrix (Bio-Rad), and used to immunoprecipitate ATR from 20 µL of egg extracts. The matrix was washed with EB and incubated in EB supplemented with 1 mM ATP, 5 mM MgCl2, and 100 µg/mL Chk1-KD in the presence or absence of various concentrations of recombinant Cut5 and claspin at 23 °C for 30 min. Each supernatant was then immunoblotted.
|
Acknowledgements |
|---|
(p60), to Noriyuki Sagata for the cDNA of Xenopus Chk1-KD, and to Yumiko Kubota for a critical reading of the manuscript. Y. Hashimoto was supported by a Research Fellowship for Young Scientists of the Japan Society for the Promotion of Science. This work was supported by Grants-in-Aid for Scientific Research on Priority Area (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan. |
Footnotes |
|---|
* Correspondence: E-mail: takisawa{at}bio.sci.osaka-u.ac.jp
|
References |
|---|
|
TopAbstractIntroductionResultsDiscussionExperimental proceduresReferences |
|---|
) in Saccharomyces cerevisiae, has a dual role in S-phase progression and at a cell cycle checkpoint. Proc. Natl. Acad. Sci. USA
92, 11791–11795.Bakkenist, C.J. & Kastan, M.B. (2004) Initiating cellular stress responses. Cell 118, 9–17.[CrossRef][Medline]
Blow, J.J. & Hodgson, B. (2002) Replication licensing—defining the proliferative state? Trends Cell. Biol. 12, 72–78.[CrossRef][Medline]
Byun, T.S., Pacek, M., Yee, M.C., Walter, J.C. & Cimprich, K.A. (2005) Functional uncoupling of MCM helicase and DNA polymerase activities activates the ATR-dependent checkpoint. Genes Dev.
19, 1040–1052.
Calzada, A., Hodgson, B., Kanemaki, M., Bueno, A. & Labib, K. (2005) Molecular anatomy and regulation of a stable replisome at a paused eukaryotic DNA replication fork. Genes Dev.
19, 1905–1919.
Chou, D.M., Petersen, P., Walter, J.C. & Walter, G. (2002) Protein phosphatase 2A regulates binding of Cdc45 to the prereplication complex. J. Biol. Chem.
277, 40520–40527.
Diffley, J.F. (2004) Regulation of early events in chromosome replication. Curr. Biol. 14, R778–R786.[CrossRef][Medline]
Furuya, K., Poitelea, M., Guo, L., Caspari, T. & Carr, A.M. (2004) Chk1 activation requires Rad9 S/TQ-site phosphorylation to promote association with C-terminal BRCT domains of Rad4TOPBP1. Genes Dev.
18, 1154–1164.
Garcia, V., Furuya, K. & Carr, A.M. (2005) Identification and functional analysis of TopBP1 and its homologs. DNA Repair 4, 1227–1239.[CrossRef][Medline]
Guo, Z., Kumagai, A., Wang, S.X. & Dunphy, W.G. (2000) Requirement for Atr in phosphorylation of Chk1 and cell cycle regulation in response to DNA replication blocks and UV-damaged DNA in Xenopus egg extracts. Genes Dev.
14, 2745–2756.
Hashimoto, Y. & Takisawa, H. (2003) Xenopus Cut5 is essential for a CDK-dependent process in the initiation of DNA replication. EMBO J. 22, 2526–2535.[CrossRef][Medline]
Hekmat-Nejad, M., You, Z., Yee, M.C., Newport, J.W. & Cimprich, K.A. (2000) Xenopus ATR is a replication-dependent chromatin-binding protein required for the DNA replication checkpoint. Curr. Biol. 10, 1565–1573.[CrossRef][Medline]
Jeong, S.Y., Kumagai, A., Lee, J. & Dunphy, W.G. (2003) Phosphorylated claspin interacts with a phosphate-binding site in the kinase domain of Chk1 during ATR-mediated activation. J. Biol. Chem.
278, 46782–46788.
Jones, R.E., Chapman, J.R., Puligilla, C., et al. (2003) XRad17 is required for the activation of XChk1 but not XCds1 during checkpoint signaling in Xenopus. Mol. Biol. Cell
14, 3898–3910.
Katou, Y., Kanoh, Y., Bando, M., et al. (2003) S-phase checkpoint proteins Tof1 and Mrc1 form a stable replication-pausing complex. Nature 424, 1078–1083.[CrossRef][Medline]
Kearsey, S.E. & Cotterill, S. (2003) Enigmatic variations: divergent modes of regulating eukaryotic DNA replication. Mol. Cell 12, 1067–1075.[CrossRef][Medline]
Kim, S.M., Kumagai, A., Lee, J. & Dunphy, W.G. (2005a) Phosphorylation of Chk1 by ATM- and Rad3-related (ATR) in Xenopus egg extracts requires binding of ATRIP to ATR but not the stable DNA-binding or coiled-coil domains of ATRIP. J. Biol. Chem.
280, 38355–38364.
Kim, S.T., Lim, D.S., Canman, C.E. & Kastan, M.B. (1999) Substrate specificities and identification of putative substrates of ATM kinase family members. J. Biol. Chem.
274, 37538–37543.
Kim, J.E., McAvoy, S.A., Smith, D.I. & Chen, J. (2005b) Human TopBP1 ensures genome integrity during normal S phase. Mol. Cell. Biol.
25, 10907–10915.
Kubota, Y. & Takisawa, H. (1993) Determination of initiation of DNA replication before and after nuclear formation in Xenopus egg cell-free extracts. J. Cell Biol.
123, 1321–1331.
Kumagai, A. & Dunphy, W.G. (2000) Claspin, a novel protein required for the activation of Chk1 during a DNA replication checkpoint response in Xenopus egg extracts. Mol. Cell 6, 839–849.[CrossRef][Medline]
Kumagai, A. & Dunphy, W.G. (2003) Repeated phosphopeptide motifs in claspin mediate the regulated binding of Chk1. Nat. Cell Biol. 5, 161–165.[CrossRef][Medline]
Kumagai, A., Guo, Z., Emami, K.H., Wang, S.X. & Dunphy, W.G. (1998) The Xenopus Chk1 protein kinase mediates a caffeine-sensitive pathway of checkpoint control in cell-free extracts. J. Cell Biol.
142, 1559–1569.
Kumagai, A., Kim, S.M. & Dunphy, W.G. (2004) Claspin and the activated form of ATR-ATRIP collaborate in the activation of Chk1. J. Biol. Chem.
279, 49599–49608.
Kumagai, A., Lee, J., Yoo, H.Y. & Dunphy, W.G. (2006) TopBP1 activates the ATR-ATRIP complex. Cell 124, 943–955.[CrossRef][Medline]
Lambert, S. & Carr, A.M. (2005) Checkpoint responses to replication fork barriers. Biochimie 87, 591–602.[Medline]
Lee, J., Kumagai, A. & Dunphy, W.G. (2003) Claspin, a Chk1-regulatory protein, monitors DNA replication on chromatin independently of RPA, ATR, and Rad17. Mol. Cell 11, 329–340.[CrossRef][Medline]
Lee, J.H. & Paull, T.T. (2005) ATM activation by DNA double-strand breaks through the Mre11–Rad50–Nbs1 complex. Science
308, 551–554.
Lupardus, P.J., Byun, T., Yee, M.C., Hekmat-Nejad, M. & Cimprich, K.A. (2002) A requirement for replication in activation of the ATR-dependent DNA damage checkpoint. Genes Dev.
16, 2327–2332.
Manke, I.A., Lowery, D.M., Nguyen, A. & Yaffe, M.B. (2003) BRCT repeats as phosphopeptide-binding modules involved in protein targeting. Science
302, 636–639.
Marheineke, K. & Hyrien, O. (2004) Control of replication origin density and firing time in Xenopus egg extracts: role of a caffeine-sensitive, ATR-dependent checkpoint. J. Biol. Chem.
279, 28071–28081.
Masumoto, H., Muramatsu, S., Kamimura, Y. & Araki, H. (2002) S-Cdk-dependent phosphorylation of Sld2 essential for chromosomal DNA replication in budding yeast. Nature 415, 651–655.[CrossRef][Medline]
Méndez, J. & Stillman, B. (2003) Perpetuating the double helix: molecular machines at eukaryotic DNA replication origins. Bioessays 25, 1158–1167.[CrossRef][Medline]
Mimura, S., Masuda, T., Matsui, T. & Takisawa, H. (2000) Central role for Cdc45 in establishing an initiation complex of DNA replication in Xenopus egg extracts. Genes Cells 5, 439–452.[Abstract]
Mimura, S. & Takisawa, H. (1998) Xenopus Cdc45-dependent loading of DNA polymerase alpha onto chromatin under the control of S-phase Cdk. EMBO J. 17, 5699–5707.[CrossRef][Medline]
Mochida, S., Esashi, F., Aono, N., Tamai, K., O’Connell, M.J. & Yanagida, M. (2004) Regulation of checkpoint kinases through dynamic interaction with Crb2. EMBO J. 28, 418–442.[CrossRef]
Nishitani, H. & Lygerou, Z. (2002) Control of DNA replication licensing in a cell cycle. Genes Cells 7, 523–534.[Abstract]
Noguchi, E., Shanahan, P., Noguchi, C. & Russell, P. (2002) CDK phosphorylation of Drc1 regulates DNA replication in fission yeast. Curr. Biol. 12, 599–605.[CrossRef][Medline]
Nyberg, K.A., Michelson, R.J., Putnam, C.W. & Weinert, T.A. (2002) Toward maintaining the genome: DNA damage and replication checkpoints. Annu. Rev. Genet. 36, 617–656.[CrossRef][Medline]
O’Connell, M.J. & Cimprich, K.A. (2005) G2 damage checkpoints: what is the turn-on?
J. Cell Sci.
118, 1–6.
Osborn, A.J. & Elledge, S.J. (2003) Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53. Genes Dev.
17, 1755–1767.
Pacek, M. & Walter, J.C. (2004) A requirement for MCM7 and Cdc45 in chromosome unwinding during eukaryotic DNA replication. EMBO J. 23, 3667–3676.[CrossRef][Medline]
Parrilla-Castellar, E.R. & Karnitz, L.M. (2003) Cut5 is required for the binding of Atr and DNA polymerase alpha to genotoxin-damaged chromatin. J. Biol. Chem.
278, 45507–45511.
Saka, Y., Esashi, F., Matsusaka, T., Mochida, S. & Yanagida, M. (1997) Damage and replication checkpoint control in fission yeast is ensured by interactions of Crb2, a protein with BRCT motif, with Cut5 and Chk1. Genes Dev.
11, 3387–3400.
Saka, Y., Fantes, P., Sutani, T., McInerny, C., Creanor, J. & Yanagida, M. (1994) Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication checkpoint control. EMBO J. 13, 5319–5329.[Medline]
Shechter, D., Costanzo, V. & Gautier, J. (2004) ATR and ATM regulate the timing of DNA replication origin firing. Nat. Cell Biol. 6, 648–655.[CrossRef][Medline]
Shimuta, K., Nakajo, N., Uto, K., Hayano, Y., Okazaki, K. & Sagata, N. (2002) Chk1 is activated transiently and targets Cdc25A for degradation at the Xenopus midblastula transition. EMBO J. 21, 3694–3703.[CrossRef][Medline]
Sogo, J.M., Lopes, M. & Foiani, M. (2002) Fork reversal and ssDNA accumulation at stalled replication forks owing to checkpoint defects. Science
297, 599–602.
St Onge, R.P., Besley, B.D., Pelley, J.L. & Davey, S. (2003) A role for the phosphorylation of hRad9 in checkpoint signaling. J. Biol. Chem.
278, 26620–26628.
Van Hatten, R.A., Tutter, A.V., Holway, A.H., Khederian, A.M., Walter, J.C. & Michael, W.M. (2002) The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication. J. Cell Biol.
159, 541–547.
Waga, S., Masuda, T., Takisawa, H. & Sugino, A. (2001) DNA polymerase is required for coordinated and efficient chromosomal DNA replication in Xenopus egg extracts. Proc. Natl. Acad. Sci. USA
98, 4978–4983.
Walter, J. & Newport, J. (2000) Initiation of eukaryotic DNA replication: origin unwinding and sequential chromatin association of Cdc45, RPA, and DNA polymerase . Mol. Cell
5, 617–627.[CrossRef][Medline]
Wang, H. & Elledge, S.J. (1999)
DRC1, DNA replication and checkpoint protein 1, functions with DPB11 to control DNA replication and the S-phase checkpoint in Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. USA
96, 3824–3829.
Wang, H. & Elledge, S.J. (2002) Genetic and physical interactions between DPB11 and DDC1 in the yeast DNA damage response pathway. Genetics
160, 1295–1304.
Yamane, K., Wu, X. & Chen, J. (2002) A DNA damage-regulated BRCT-containing protein, TopBP1, is required for cell survival. Mol. Cell. Biol.
22, 555–566.
Yan, S., Lindsay, H.D. & Michael, W.M. (2006) Direct requirement for Xmus101 in ATR-mediated phosphorylation of Claspin bound Chk1 during checkpoint signaling. J. Cell Biol.
173, 181–186.
You, Z., Kong, L. & Newport, J. (2002) The role of single-stranded DNA and polymerase alpha in establishing the ATR, Hus1 DNA replication checkpoint. J. Biol. Chem.
277, 27088–27093.
Yu, X., Chini, C.C., He, M., Mer, G. & Chen, J. (2003) The BRCT domain is a phospho-protein binding domain. Science
302, 639–642.
Zou, L., Cortez, D. & Elledge, S.J. (2002) Regulation of ATR substrate selection by Rad17-dependent loading of Rad9 complexes onto chromatin. Genes Dev.
16, 198–208.
Zou, L. & Elledge, S.J. (2003) Sensing DNA damage through ATRIP recognition of RPA-ssDNA complexes. Science
300, 1542–1548.
Received: 19 March 2006
Accepted: 25 May 2006
This article has been cited by other articles:
|
|
 |
|
  A. Chowdhury, G. Liu, M. Kemp, X. Chen, N. Katrangi, S. Myers, M. Ghosh, J. Yao, Y. Gao, P. Bubulya, et al. The DNA Unwinding Element Binding Protein DUE-B Interacts with Cdc45 in Preinitiation Complex Formation Mol. Cell. Biol., March 15, 2010; 30(6): 1495 - 1507. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. Lee and W. G. Dunphy Rad17 Plays a Central Role in Establishment of the Interaction between TopBP1 and the Rad9-Hus1-Rad1 Complex at Stalled Replication Forks Mol. Biol. Cell, March 15, 2010; 21(6): 926 - 935. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 K. Yoshida, N. Sugimoto, S. Iwahori, T. Yugawa, M. Narisawa-Saito, T. Kiyono, and M. Fujita CDC6 interaction with ATR regulates activation of a replication checkpoint in higher eukaryotic cells J. Cell Sci., January 15, 2010; 123(2): 225 - 235. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. L. Sansam, N. M. Cruz, P. S. Danielian, A. Amsterdam, M. L. Lau, N. Hopkins, and J. A. Lees A vertebrate gene, ticrr, is an essential checkpoint and replication regulator Genes & Dev., January 15, 2010; 24(2): 183 - 194. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 L. A. Lindsey-Boltz, O. Sercin, J.-H. Choi, and A. Sancar Reconstitution of Human Claspin-mediated Phosphorylation of Chk1 by the ATR (Ataxia Telangiectasia-mutated and Rad3-related) Checkpoint Kinase J. Biol. Chem., November 27, 2009; 284(48): 33107 - 33114. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Tanaka, Y. Kubota, T. Tsujimura, M. Kumano, H. Masai, and H. Takisawa Replisome progression complex links DNA replication to sister chromatid cohesion in Xenopus egg extracts Genes Cells, August 1, 2009; 14(8): 949 - 963. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. Yoo, A. Kumagai, A. Shevchenko, A. Shevchenko, and W. G. Dunphy The Mre11-Rad50-Nbs1 Complex Mediates Activation of TopBP1 by ATM Mol. Biol. Cell, May 1, 2009; 20(9): 2351 - 2360. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-H. Choi, L. A. Lindsey-Boltz, and A. Sancar Cooperative activation of the ATR checkpoint kinase by TopBP1 and damaged DNA Nucleic Acids Res., April 1, 2009; 37(5): 1501 - 1509. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Yan and W. M. Michael TopBP1 and DNA polymerase-{alpha} directly recruit the 9-1-1 complex to stalled DNA replication forks J. Cell Biol., March 23, 2009; 184(6): 793 - 804. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
B. Shiotani and L. Zou ATR signaling at a glance J. Cell Sci., February 1, 2009; 122(3): 301 - 304. [Full Text] [PDF]
|
|
|
|
|
|
F. Puddu, M. Granata, L. Di Nola, A. Balestrini, G. Piergiovanni, F. Lazzaro, M. Giannattasio, P. Plevani, and M. Muzi-Falconi Phosphorylation of the Budding Yeast 9-1-1 Complex Is Required for Dpb11 Function in the Full Activation of the UV-Induced DNA Damage Checkpoint Mol. Cell. Biol., August 1, 2008; 28(15): 4782 - 4793. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Mordes, G. G. Glick, R. Zhao, and D. Cortez TopBP1 activates ATR through ATRIP and a PIKK regulatory domain Genes & Dev., June 1, 2008; 22(11): 1478 - 1489. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Lee, A. Kumagai, and W. G. Dunphy The Rad9-Hus1-Rad1 Checkpoint Clamp Regulates Interaction of TopBP1 with ATR J. Biol. Chem., September 21, 2007; 282(38): 28036 - 28044. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-H. Choi, L. A. Lindsey-Boltz, and A. Sancar Reconstitution of a human ATR-mediated checkpoint response to damaged DNA PNAS, August 14, 2007; 104(33): 13301 - 13306. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
H. Y. Yoo, A. Kumagai, A. Shevchenko, A. Shevchenko, and W. G. Dunphy Ataxia-telangiectasia Mutated (ATM)-dependent Activation of ATR Occurs through Phosphorylation of TopBP1 by ATM J. Biol. Chem., June 15, 2007; 282(24): 17501 - 17506. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Y. Jeon, K. Y. Lee, M. J. Ko, Y. S. Lee, S. Kang, and D. S. Hwang Human TopBP1 Participates in Cyclin E/CDK2 Activation and Preinitiation Complex Assembly during G1/S Transition J. Biol. Chem., May 18, 2007; 282(20): 14882 - 14890. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. Zou Single- and double-stranded DNA: building a trigger of ATR-mediated DNA damage response Genes & Dev., April 15, 2007; 21(8): 879 - 885. [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | ADVANCED SEARCH | TABLE OF CONTENTS |
