Genetic and Physical Interactions Between DPB11 and DDC1 in the Yeast DNA Damage Response Pathway

Corresponding author: Stephen J. Elledge, Baylor College of Medicine, 1 Baylor Plaza, Houston, TX 77030., selledge{at}bcm.tmc.edu (E-mail)

Communicating editor: A. P. MITCHELL

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DPB11 is essential for DNA replication and S/M checkpoint control in Saccharomyces cerevisiae. The Dpb11 protein contains four BRCT domains, which have been proposed to be involved in protein-protein interactions. To further investigate the regulation and function of Dpb11, a yeast two-hybrid screen was carried out to identify proteins that physically interact with Dpb11. One positive clone isolated from the screen encoded a carboxyl-terminal fragment of Ddc1 (339–612 aa). Ddc1 is a DNA damage checkpoint protein, which, together with Mec3 and Rad17, has been proposed to form a PCNA-like complex and acts upstream in the DNA damage checkpoint pathways. We further determined that the carboxyl region of Dpb11 is required for its interaction with Ddc1. DDC1 and DPB11 also interact genetically. The ddc1 dpb11-1 double mutant is more UV and MMS sensitive than the ddc1 or the dpb11-1 single mutants. Furthermore, the double mutant is more hydroxyurea sensitive and displayed a lower restrictive temperature than dpb11-1. These results suggest that DPB11 and DDC1 may function in the same or parallel pathways after DNA damage and that DDC1 may play a role in responding to replication defects.

IN the budding yeast Saccharomyces cerevisiae, several DNA replication proteins have been shown to be essential for S/M checkpoint control, which inhibits mitotic entry before DNA replication during S-phase is completed (ELLEDGE 1996 ; LOWNDES and MURGUIA 2000 ). In S. cerevisiae, the S/M checkpoint is assayed by examining the ability of cells to undergo anaphase-like spindle elongation when DNA replication is blocked by hydroxyurea (HU). Pol2, the catalytic subunit of DNA polymerase II (), is required for the S/M checkpoint, perhaps by acting as a sensor of DNA replication blocks (NAVAS et al. 1995 ). Dpb11 physically interacts with Pol2 and is responsible for recruiting Pol2 to DNA replication origins (MASUMOTO et al. 2000 ). Like Pol2, Dpb11 is also required for the S/M checkpoint (ARAKI et al. 1995 ; WANG and ELLEDGE 1999 ). Dpb11 associates with Drc1 (Sld2), another protein required for both DNA replication and S/M checkpoint control (KAMIMURA et al. 1998 ; WANG and ELLEDGE 1999 ).

Dpb11 is an evolutionarily conserved protein. Cut5 in Schizosaccharomyces pombe (SAKA and YANAGIDA 1993 ; SAKA et al. 1994A , SAKA et al. 1994B ), mus101 in Drosophila (YAMAMOTO et al. 2000 ), and human TopBP1 (YAMANE et al. 1997 ; YAMANE and TSURUO 1999 ; MAKINIEMI et al. 2001 ) show sequence and functional similarity to Dpb11. All of them have been shown to be required for DNA replication and with the exception of mus101 are also essential for the S/M checkpoint control. Moreover, TopBP1, like Dpb11, also interacts with DNA polymerase (MAKINIEMI et al. 2001 ).

Dpb11 and its homologs contain BRCA1 carboxy-terminal (BRCT) domains, a putative protein-protein interaction motif (BORK et al. 1997 ; HUYTON et al. 2000 ). BRCT domains have been identified in >50 proteins involved in DNA repair, recombination, or cell cycle control. X-ray crystallography revealed the three-dimensional structure of the BRCT domain of XRCC1 as a globular motif (ZHANG et al. 1998 ). Accumulating evidence suggests that BRCT domains mediate homo/hetero BRCT multimer formation, non-BRCT interactions, and DNA end binding (HUYTON et al. 2000 ). For example, XRCC1 forms a heterodimer via its BRCT domain with DNA ligase III (TAYLOR et al. 1998 ). Rad9, a DNA damage checkpoint protein in S. cerevisiae, oligomerizes after DNA damage through its BRCT domain (SOULIER and LOWNDES 1999 ). A yeast two-hybrid screen with S. pombe Cut5 led to the identification of the Crb2 protein, which is also a BRCT domain-containing checkpoint protein (SAKA et al. 1997 ). Recently, it has been shown that TopBP1 interacts with the checkpoint protein hRad9 through its BRCT domains (MAKINIEMI et al. 2001 ).

To fully understand the function of Dpb11 and to study the mechanism of the S/M checkpoint pathway, we carried out a yeast two-hybrid screen for proteins that physically interact with Dpb11. One of the putative Dpb11 interacting clones encoded the carboxyl terminus of a DNA damage checkpoint protein, Ddc1. We focused our study on Ddc1 because the S. pombe homolog of Dpb11, Cut5, was shown to be required for DNA damage checkpoint control (MCFARLANE et al. 1997 ; VERKADE and O'CONNELL 1998 ). Therefore we wished to explore the potential role of Dpb11 in response to DNA damage in the context of DDC1. Moreover, the function of DDC1 has been established to some extent. DDC1 belongs to the MEC3, RAD17, RAD24 epistasis group, which, together with RAD9, is proposed to act at the beginning of the DNA damage checkpoint pathways (KONDO et al. 1999 ). However, ddc1 mutants are competent for the S/M checkpoint, although DDC1 is required for slowing down DNA replication in the presence of DNA damage (LONGHESE et al. 1997 ), also known as the intra-S-phase checkpoint. Ddc1, Mec3, and Rad17 have been proposed to form a proliferating cell nuclear antigen (PCNA)-like complex. PCNA functions as the sliding clamp that tethers DNA polymerase to its DNA template (KONDO et al. 1999 ; VENCLOVAS and THELEN 2000 ). On the basis of this structural similarity, it is proposed that the Ddc1-Mec3-Rad17 complex serves as a structural mediator for initiation of checkpoint signaling and provides processivity for DNA repair proteins (VENCLOVAS and THELEN 2000 ).

We showed here that Ddc1 and Dpb11 not only physically interact, but they also genetically interact with each other. The dpb11 ddc1 double mutant is more sensitive to DNA damaging agents and DNA replication inhibitors, suggesting that Dpb11 and Ddc1 might collaborate in responding to DNA abnormalities. Deletion of DDC1 also lowers the restrictive temperature of the dpb11 mutant, implying that DDC1 is required for monitoring any DNA replication defects or DNA damage resulting from dpb11 mutation.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

DNA plasmids:
pHW1 (pAS2-DPB11) was constructed by first engineering a NdeI site at the start codon of DPB11 and then subcloning the entire DPB11 coding sequence from NdeI (-3 bp) to SalI (2500 bp) into the NdeI/SalI site of the bait vector, pAS2 (BAI and ELLEDGE 1997 ). DPB11 coding sequence was cloned into the SmaI/SacI site of pBAD98 (DESANY et al. 1998 ), generating pHW82 (pBAD98-DPB11). The HindIII/SacI fragment containing the DPB11 coding region from pHW82 was transferred to the HindIII/SacI site of pRS415 (SIKORSKI and HIETER 1989 ), generating pHW84. The promoter region of DPB11, starting from a HindIII site (-680 bp), was amplified by PCR and a NdeI site was engineered just at the start codon of DPB11. The resulting PCR product was digested by HindIII and NdeI and cloned into the HindIII/NdeI site of pHW84, generating pHW85 (pRS415-DPB11 under its own promoter).

Yeast strains:
Yeast strains used in this study are isogenic with the W303-derived Y300 strain. All derived strains were constructed using standard genetic crosses and are listed in Table 1. Gene disruptions were performed by replacing one copy of the target gene from a diploid wild-type genome with the HIS3 marker (LORENZ et al. 1995 ) and the correct targeting events were confirmed by Southern blotting analysis. Y1187 containing the hemagglutinin (HA)-tagged Ddc1 was obtained by integrating PstI-cleaved pML119 (LONGHESE et al. 1997 ) into Y300.

Yeast two-hybrid screen:
The yeast two-hybrid screen was performed as described (BAI and ELLEDGE 1997 ). Basically, Y190 was sequentially transformed with pAS2-DPB11 (pHW1) and a S. cerevisiae cDNA-GAL4 activation domain (AD) fusion library. An estimated 1 million transformants were screened. Yeast clones containing potential Dpb11 interacting proteins were identified by growth on SC-Trp, Leu, His plates with 50 mM 3-amino-1,2,4-triazole (3-AT) (A8056; Sigma, St. Louis) for HIS3 transcription. A total of 48 clones were obtained from HIS3 selection and 12 of them also turned blue by X-gal colony filter assay for LacZ transcription. To eliminate false-positive clones, all the positive clones were transformed back into Y190 with either pAS2 empty vector or other bait plasmids encoding Cdk2, Snf1, lamin, or p53, respectively (BAI and ELLEDGE 1997 ). All of these combinations did not lead to the activation of either the HIS3 or LacZ reporter gene.

Construction of temperature-sensitive or HU-sensitive dpb11 mutants:
pHW85 (pRS415-DPB11) was used as template to carry out the low-fidelity PCR reaction (WANG and ELLEDGE 1999 ). Primers used in the mutagenesis PCR are as follows: Dpb11-1-1, 5'-CTTCTATTTCTAGTATGGCAGG-3' (upstream of DPB11 coding sequence); and Drc1-9, 5'-GTGAGTTACCTCACTCATTAGGC-3' (pRS415 vector sequence; WANG and ELLEDGE 1999 ). The DPB11-mN library was generated by replacing the NdeI/PstI fragment of pHW85 (including the N-terminal 770 bp of DPB11) by the PCR products and the DPB11-mC library was generated by replacing the PstI/SacI fragment of pHW85 (including C-terminal 1.5 kb of Dpb11 coding sequence) by the PCR products.

The two libraries were screened in YHW186 as described (WANG and ELLEDGE 1999 ). No temperature-sensitive mutants were isolated from the DPB11-mC library. Seven dpb11 temperature-sensitive (ts) alleles, called pHW164 (dpb11-9), pHW165 (dpb11-13), pHW166 (dpb11-3), pHW167 (dpb11-2), pHW168 (dpb11-11), pHW169 (dpb11-4), and pHW170 (dpb11-12), were isolated from the DPB11-mN library. Three of these were integrated into yeast strains, generating Y1198 (dpb11-2, ts at 34°), Y1199 (dpb11-3, ts at 32°), and Y1200 (dpb11-4, ts at 30°). Four new HU-sensitive dpb11 alleles were isolated, two from the DPB11-mN library and two from the DPB11-mC library. They were named pHW160 (dpb11-5), pHW161 (dpb11-6), pHW 162 (dpb11-7), and pHW163 (dpb11-8).

UV sensitivity measurement:
Approximately 500 log-phase cells were spread on plates and then treated with different doses of UV light. UV sensitivity was measured by counting the colonies formed after several days.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dpb11 interacts with the C terminus of Ddc1:
Dpb11 contains four BRCT domains that are likely to mediate protein-protein interactions. To explore possible Dpb11- binding proteins, we carried out a yeast two-hybrid screen for Dpb11. The bait plasmid, pAS2-DPB11, was constructed by fusing the entire Dpb11 protein to the C terminus of the GAL4 DNA-binding (DB) domain. We first confirmed that the fusion protein encoded by pAS2-DPB11 is properly expressed and functions as wild-type Dpb11 because it could complement the growth of dpb11 null cells (Fig 1A).

View larger version (32K): In this window In a new window Download PPT slide   Figure 1. Dpb11 and Ddc1 interact with each other through their C termini. (A) pAS2-DPB11, which encodes the GAL4 DNA-binding domain-Dpb11 fusion protein, can suppress the lethality of dpb11. Y1186, a dpb11 null strain containing a pHW82 (pBAD98-DPB11, URA3) plasmid, was transformed with pAS2 or pAS2-DPB11 and then streaked on SC-Trp or SC-Trp plates containing 5-fluoroorotic acid (5-FOA). pAS2-DPB11 could support this strain to grow on a 5-FOA plate. (B) The C terminus of Ddc1 (339–612 aa) interacts with Dpb11 in the yeast two-hybrid system. Strain Y190 carrying the plasmids encoding the bait and/or prey proteins as indicated was incubated on SC-Trp, Leu, His plates containing 15 mM 3-AT to test for activation of the HIS3 reporter and the colonies were also tested for LacZ transcription by X-gal assay. (C and D) The C-terminal region of Dpb11 is required for interaction with Ddc1. Strain Y190 was transformed with the constructs indicated in C. Protein-protein interaction was assessed by the growth of transformants on 15 mM 3-AT and by X-gal assays. The results are listed in D.

A S. cerevisiae cDNA GAL4AD fusion library was screened. Twelve clones were isolated from an estimated 1 million transformants as positive for the reporter gene activity (His+ LacZ+). Two out of the 12 positive clones encoded the C-terminal 274 residues of Ddc1, named Ddc1-C (Fig 1B).

The C-terminal region of Dpb11 is responsible for its interaction with Ddc1:
To identify the region of Dpb11 that is responsible for its interaction with Ddc1, two truncated forms of Dpb11 were fused to the GAL4-DB domain. Each contains two BRCT domains and they are named Dpb11-N [1–256 amino acids (aa)] and Dpb11-C (251–764 aa), respectively (Fig 1C). By examining the activation of the reporter genes, we found that Dpb11-C, but not Dpb11-N, interacted with Ddc1. Although no interaction between full-length Dpb11 and Ddc1 could be detected, Dpb11-C interacts with both full-length Ddc1 and Ddc1-C. Interestingly, dpb11-1, which encodes a C-terminal truncated protein (KAMIMURA et al. 1998 ), is also defective in interacting with Ddc1. This result further supported the interpretation that the C terminus of Dpb11 is responsible for its interaction with Ddc1.

Dpb11 and Ddc1 physically interact with each other in vitro:
To test if we could detect a physical association between Dpb11 and Ddc1, we tried both in vivo and in vitro methods. We were unable to co-immunoprecipitate these proteins in vivo in untreated, methyl methane sulfonate (MMS)-, or HU-treated cells. We reasoned that their association might be too weak to survive immunoprecipitation conditions or it may occur on chromatin. Thus we tested whether Dpb11 and Ddc1 interact with each other in vitro. An in vitro glutathione S-transferase (GST) pull-down experiment was performed. GST-Dpb11, but not GST, could bind HA-tagged Ddc1 from the yeast extract (Fig 2A), indicating that Dpb11 interacts with Ddc1 in vitro.

View larger version (45K): In this window In a new window Download PPT slide   Figure 2. (A) Ddc1 interacts with Dpb11 in vitro. GST or GST-Dpb11 was purified from baculovirus-infected insect cells using glutathione beads. Approximately 0.5 µg of purified proteins bound on glutathione beads was incubated with 400 µg of yeast protein extract from Y1187 (DDC1-HA) at 4° for 2 hr. Beads were washed and protein samples were analyzed by Western blotting. Ddc1 was detected using anti-HA antibodies. (B) Overexpression of DDC1 in drc1-1 is toxic and this toxicity can be reversed by co-overexpression of DPB11. drc1-1 (Y799) cells were transformed with vector alone or pGAL-DDC1 (pML109; LONGHESE et al. 1997 ). The transformants were grown on either glucose or galactose plates as indicated and incubated at 24° (top). drc1-1 carrying pGAL-DDC1 were transformed with pGAL-DPB11 and the growth of the transformants was tested on either glucose or galactose plates as indicated (bottom).

If Ddc1 physically interacts with Dpb11, we would expect genetic interactions between them as well. Therefore, we examined interactions between mutations of DDC1 and DPB11 and other components of the DPB11 pathway. It has been shown that Dpb11 physically interacts with Drc1, another DNA replication and S/M checkpoint protein (KAMIMURA et al. 1998 ; WANG and ELLEDGE 1999 ). We observed that overexpression of Ddc1 is toxic to drc1-1 mutants and this toxicity can be reversed when DPB11 is co-overexpressed (Fig 2B). The toxicity of overexpression of DDC1 is specific to drc1-1 cells. DDC1 overexpression does not confer toxicity in wild-type or dpb11-1 cells (data not shown). Thus, the toxicity is not general. A plausible explanation for these observations is that Dpb11 interacts with both Ddc1 and Drc1 and overexpressed Ddc1 competes with Drc1 for Dpb11, therefore resulting in toxicity in drc1-1. Co-overexpression of DPB11 alleviates this competition and relieves the toxicity.

Genetic interactions between DPB11 and DDC1:
Ddc1 is essential for DNA damage checkpoint control and a ddc1 mutant is very sensitive to UV irradiation (LONGHESE et al. 1997 ). dpb11-1 mutants have also been shown to be slightly sensitive to UV (ARAKI et al. 1995 ). We found that the ddc1 dpb11 double mutant is more UV and MMS sensitive than either single mutant (Fig 3A). To test if DPB11 and DDC1 share some redundant functions and might mutually suppress the defects of each other, we overexpressed DPB11 in ddc1 mutants and vice versa. No suppression of the UV sensitivity of ddc1 was observed with overexpressed DPB11 (data not shown). When DDC1 was overexpressed in dpb11-1 cells, a partial suppression of the UV sensitivity of dpb11-1 mutants was observed (Fig 3B), suggesting that DPB11 and DDC1 function in the same or parallel pathways in response to DNA damage.

View larger version (13K): In this window In a new window Download PPT slide   Figure 3. Genetic interactions between DPB11 and DDC1. (A) ddc1 dpb11-1 double mutants showed enhanced UV-sensitive and MMS-sensitive phenotype. Log-phase () wild-type (Y300), () ddc1 (Y1135), (•) dpb11-1 (Y1185), and () ddc1 dpb11-1 (Y1189) cells were spread on YPD plates and UV irradiated at different doses. Viability was scored by counting the colonies that grew up on the plates after 3 days at 24° (top). Log-phase wild-type (Y300), ddc1 (Y1135), dpb11-1 (Y1185), and ddc1 dpb11-1 (Y1189) cells were treated with 0.01% MMS. Aliquots were withdrawn at the indicated times to test viability (bottom). (B) Overexpression of DDC1 slightly suppresses the UV sensitivity of dpb11-1 cells. (•, ) Wild-type (Y300), (, ) dpb11-1 (Y1185), and (, ) ddc1 (Y1135) cells, harboring either empty vector (solid) or GAL-DDC1 (open), were cultured to log phase in galactose medium. Cells were spread on SC-Ura, Gal plates and treated with different doses of UV light and cell viability was determined as in A.

Dpb11 is not essential for the DNA-damage-induced hyperphosphorylation of Ddc1:
Ddc1 is hyperphosphorylated after DNA damage in a MEC1-dependent manner (LONGHESE et al. 1997 ; PACIOTTI et al. 1998 ). To test if Dpb11 is required for the phosphorylation of Ddc1 after DNA damage, wild-type and dpb11-1 cells were treated with UV (50 J/m²), and then the extent of Ddc1 phosphorylation was examined by Western blot. However, Ddc1 phosphorylation in dpb11-1 mutants was not reduced compared to that in wild-type cells, suggesting that Dpb11 is not essential for DNA damage-induced Ddc1 phosphorylation and is not functioning upstream in that capacity (data not shown).

dpb11-1 mutants are proficient for the G2/M DNA damage checkpoint:
The genetic interaction between DPB11 and DDC1 suggests that Dpb11 might play a role in response to DNA damage. Pol2 has been shown to be important for the DNA damage checkpoint control in S-phase cells, while Rad9 mainly functions in cells outside of S-phase (NAVAS et al. 1996 ). It is possible that Dpb11 is also essential for DNA damage checkpoint control in S-phase only. However, when -factor-arrested or nocodazole-arrested dpb11-1 cells were irradiated by UV light, they were still more sensitive to UV than to wild-type cells (Fig 4A), suggesting that Dpb11 also functions outside of S-phase.

View larger version (24K): In this window In a new window Download PPT slide   Figure 4. dpb11-1 is proficient for the G2/M DNA damage checkpoint. (A) G1- and G2-arrested dpb11-1 cells are UV sensitive. -factor or nocodazole-arrested wild-type (Y300) and dpb11-1 (Y1185) cells were cultured on YPD plates and UV irradiated at indicated doses. (B) Log-phase wild-type (Y300), dpb11-1 (Y1185), and ddc1 (Y1135) cells were arrested with 10 µg/ml nocodazole and irradiated with 50 J/m2 UV. At the indicated times, the percentages of uninucleate large-budded cells were scored by 4',6-diamidino-2-phenylindole staining.

S. pombe Cut5 has been implicated in DNA damage checkpoint control. A role in controlling cell cycle transitions after DNA damage could explain the UV sensitivity of dpb11-1 in G2. To test this, wild-type, ddc1, and dpb11-1 cells were arrested in G2 by nocodazole treatment and shifted to 36° for 30 min to inactivate the dpb11-1 mutant. Then, the cells were irradiated with UV and released at 36°. The percentage of cells that had one nucleus was counted to monitor the anaphase entry. As reported previously (LONGHESE et al. 1997 ), ddc1 is DNA damage checkpoint defective, as 40% of cells entered anaphase in the presence of DNA damage. In contrast, dpb11-1 mutant cells behaved like wild-type cells and maintained cell cycle arrest. Therefore, they are proficient for cell cycle arrest after DNA damage, and their UV-sensitivity phenotype during G2 is more likely to result from a DNA repair defect.

DDC1 plays a role in response to DNA replication defects:
In experiments designed to examine genetic interactions between DPB11 and DDC1 in response to S-phase stress, we observed that double mutants between the DDC1 group of genes (DDC1, MEC3, and RAD17) and dpb11-1 are much more sensitive to HU than either of the single mutants. In contrast to the DDC1 group of genes, rad9 dpb11-1 double mutants did not have a dramatic additive HU-sensitive phenotype (Fig 5). This observation suggested that DDC1 might play a role in response to DNA replication defects.

View larger version (92K): In this window In a new window Download PPT slide   Figure 5. Deletion of DDC1, MEC3, and RAD17, but not RAD9, makes dpb11-1 more HU sensitive. Cells of the indicated genotypes were cultured to log phase, and then 10-fold serial dilutions of cells were spotted onto either YPD plates or YPD with 0.1 M HU and incubated at 24° for several days.

In addition, mutations in DDC1 lowered the restrictive temperature of dpb11-1 (Fig 6), indicating that DNA damage checkpoint or some aspect of Ddc1 function is required for the survival of dpb11-1 at higher temperatures. Since DDC1 has not been shown to be involved in either DNA replication or the S/M checkpoint, it is possible that the defects of dpb11-1 introduce DNA damage during S-phase, which requires the DNA damage checkpoint response pathway. If this is the case, then proteins involved in DNA damage repair will also be required for dpb11-1's survival. Double mutants between dpb11 and rad51 or xrs2, DNA damage repair mutants, were constructed and observed for an exacerbated phenotype. The dpb11 xrs2 double mutants did have a lower restrictive temperature than dpb11-1, but the dpb11 rad51 mutants did not (Fig 6 and data not shown). However, in addition to its DNA damage repair function, XRS2 has also been shown to be important in some aspect of checkpoint control (D'AMOURS and JACKSON 2001 ; GRENON et al. 2001 ; USUI et al. 2001 ). Therefore, the genetic interactions we observed between DPB11 and XRS2 might also be due to the defective checkpoint control in xrs2 cells.

View larger version (59K): In this window In a new window Download PPT slide   Figure 6. DNA damage checkpoint genes and DNA damage repair genes are required for the survival of dpb11-1 at higher temperature. (A) Cells of the indicated genotypes were cultured to log phase, and then 10-fold serial dilutions of cells were spotted onto YPD plates and incubated at the indicated temperatures. (B) Double mutants between ddc1 and different dpb11 alleles are more temperature sensitive than dpb11 single mutants.

Novel alleles of DBP11 reveal a linkage between DNA replication defects and S/M checkpoint defects:
We wished to determine whether the genetic interactions we observed between dpb11-1 and ddc1 mutants were allele specific or reflected a general need for DDC1 function in response to an absence of DPB11 function. However, there was only one allele of DBP11, dpb11-1, which is both ts and HU sensitive. Since DPB11 functions in both DNA replication and the S/M checkpoint pathways in S. cerevisiae and the dpb11-1 allele is defective for both of these functions, it was unclear which of these defects needed DDC1 function. We thus carried out a screen for additional dpb11 alleles, trying to separate the two functions of Dpb11 by mutation.

We independently mutagenized the N terminus and the C terminus of Dpb11 and the resulting mutagenized libraries, DPB11-mN and DPB11-mC, were screened for either ts or HU-sensitive dpb11 mutants (see MATERIALS AND METHODS). Four HU-sensitive alleles were isolated with similar HU sensitivity as dpb11-1; however, all were also ts (data not shown). Seven new ts alleles of dpb11 were isolated when the DPB11-mN library was used and all were HU sensitive. Three of them were integrated into the genome. Interestingly, all three new ts alleles, dpb11-2, dpb11-3, and dpb11-4, elongated their spindles like dpb11-1 after 2 hr when cultured at 37°. Furthermore, all three mutants lost 90% viability after 4 hr at 37° (data not shown). These results suggested that they are also defective in the S/M checkpoint.

We mapped the mutation sites for all the dpb11-1 alleles we isolated (Table 2). However, no common residues were mutated in these mutants. Several of the mutated amino acids are conserved residues in the BRCT domains and some are conserved between Dpb11 and Cut5. It is possible that the mutations at those sites structurally interfere with the function of Dpb11.

We were unable to obtain specific S/M checkpoint-defective or DNA replication-defective dpb11-1 mutant alleles, indicating that unlike Pol2, the DNA replication function and S/M checkpoint function of Dpb11 are unlikely to be separated by mutation.

The genetic interactions between DPB11 and DDC1 are not allele specific:
With three new ts alleles available, we then examined whether the genetic interactions between DPB11 and DDC1 are allele specific by crossing dpb11-2, dpb11-3, and dpb11-4 mutants with ddc1 mutants. The resulting double mutants were each more temperature sensitive than dpb11 single mutants, indicating that the interaction between these two genes is not allele specific and reflects a general defect common to each dpb11 allele (Fig 6).

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Dpb11 is an essential gene that is required for both DNA replication and S/M checkpoint controls (ARAKI et al. 1995 ; WANG and ELLEDGE 1999 ). The fact that we failed to separate these two functions of Dpb11 by mutation strongly suggests that the two functions are connected with each other. Our studies argue against the model that Dpb11 has two domains that have separate and independent functions; instead, it is likely that the S/M checkpoint function of Dpb11 is directly linked to its DNA replication function. In addition, as a number of other proteins [Drc1 (Sld2), Orc1, and Rfc1] involved in initiation of DNA replication have S/M checkpoint defects when treated with HU, it is likely that the deficiency responsible for this is their defect in DNA replication. One could envision a model in which fewer active replication forks give rise to a proportionally lower checkpoint signal. Once the level of DNA replication drops below the threshold needed to activate enough Rad53 in response to HU to arrest the cell cycle, a checkpoint defect occurs. In this threshold model, these initiator proteins would have only indirect roles in transducing the replication stress signals.

Dpb11 contains four BRCT domains that are believed to be important for mediating protein-protein interaction. In an attempt to identify proteins that physically interact with Dpb11, we carried out a yeast two-hybrid screen. One of the positive clones encodes the C terminus of the DNA damage checkpoint protein Ddc1 (LONGHESE et al. 1997 ). Ddc1 interacts with the C terminus of Dpb11 containing the third and fourth BRCT repeats. Interestingly, the dpb11-1 mutation, which encodes a C-terminal truncated form of the Dpb11 protein, is defective for the interaction with Ddc1, further supporting the notion that the C terminus of Dpb11 is important for its interaction with Ddc1. We failed to detect an interaction between Dpb11 and Ddc1 by co-immunoprecipitation experiments. Since Dpb11 interacts with Ddc1 in vitro, we speculate that the in vivo interaction between Dpb11 and Ddc1 is normally regulated and their interaction may occur only transiently under special circumstances, such as on chromatin at a stalled replication fork.

What is the significance of the interaction between Dpb11 and Ddc1? One possibility is that DPB11 plays a role in response to DNA damage, where it utilizes Ddc1 in some capacity. dpb11-1 mutants are sensitive to various DNA damaging agents, such as UV and MMS, even outside of S-phase. However, we did not observe cell cycle arrest defects in dpb11-1 mutants after DNA damage and there is no significant additive phenotype in terms of activation of Rad53 phosphorylation in dpb11-1 ddc1 double mutants after DNA damage (data not shown). These results indicate that Dpb11 is not essential for DNA damage checkpoint signaling. The S. pombe homolog of Cut5 has been shown to be required for DNA damage checkpoint signaling (MCFARLANE et al. 1997 ; VERKADE and O'CONNELL 1998 ). If DPB11 plays a role in DNA damage checkpoint signaling, it is likely to be minor and is unlikely to explain DBP11's DNA damage sensitivity.

Our data and other published reports suggest that Dpb11 might be involved in DNA repair. This is supported by several lines of evidence. First, Dpb11 physically interacts with Pol2, the catalytic subunit of DNA polymerase (MASUMOTO et al. 2000 ). Pol2 has been shown to be involved in nucleotide excision repair, DNA double-strand break repair, and other types of repair (WANG et al. 1993 ; ABOUSSEKHRA et al. 1995 ; BUDD and CAMPBELL 1995 ; KRAMATA et al. 1998 ; HOLMES and HABER 1999 ). Biochemical studies also indicate that Dpb11 is responsible for recruiting Pol2 to origins during DNA replication (MASUMOTO et al. 2000 ). Thus, it is possible that Dpb11 collaborates with Pol2 during the DNA damage repair process or potentially recruits Pol2 to sites of damage. Second, our studies indicated strong genetic interactions between DPB11 and DNA damage checkpoint genes. Since the dpb11 mutant appears proficient for arresting the cell cycle after DNA damage, dpb11-1's DNA damage sensitivity suggests it is likely to be involved in the damage repair process. Finally, it has been shown that the human homolog of Dpb11, TopBP1, binds DNA breaks in vitro (YAMANE and TSURUO 1999 ) and co-localizes with Brca1 after DNA damage in vivo (MAKINIEMI et al. 2001 ), suggesting a role for TopBP1, and indirectly for Dpb11, in DNA repair. Interestingly, TopBP1 also physically interacts with hRad9, the human homolog of Ddc1 (MAKINIEMI et al. 2001 ). It has been proposed that after DNA damage, the Ddc1/Mec3/Rad17 complex, in addition to sensing the damage signal, might also recruit DNA damage repair proteins to the sites of DNA damage. Therefore, given the finding that Ddc1 interacts with Dpb11, it is possible that Dpb11 is one of the repair proteins that is recruited by Ddc1.

Another explanation for the Ddc1-Dpb11 interaction is that Ddc1 might collaborate with Dpb11 in response to DNA replication defects. Although DDC1 is not essential for DNA replication, it was identified from a synthetic lethal screen with a primase subunit mutant, suggesting Ddc1 might have a role in monitoring DNA replication. Consistent with this model, ddc1 dpb11-1 double mutants are much more HU sensitive than either single mutant. This is true for other mutants in the DDC1 epistasis group such as rad17, rad24, and mec3. The need for DDC1 could be explained if HU treatment of dpb11 mutants induced DNA damage and therefore required DDC1's role in checkpoint control. However, rad9 mutants, which are equally defective in DNA damage checkpoint control as ddc1 mutants, do not enhance the HU sensitivity of dpb11 mutants, which suggests that it is not the checkpoint function of DDC1 that is required for survival of dpb11 mutants experiencing replication stress. Instead these results argue that Ddc1 also plays a role after DNA replication is compromised, and this role is not related to cell cycle arrest. Therefore, we suspect that DDC1 is playing a role in some aspect of repair at disrupted replication forks. The Ddc1-Mec3-Rad17 complex has previously been implicated in DNA repair because rates of excision of DNA from telomere regions in cdc13 mutants are significantly reduced in mec3 and rad17 mutants (LYDALL and WEINERT 1995 ), suggesting they might recruit nucleases to telomeric regions of DNA in the absence of Cdc13 function. Recruitment of nucleases or other repair proteins such as helicases could facilitate the repair of damaged replication forks.

In contrast to the case of DNA replicational stress, both DDC1 and RAD9 are required for the survival of dpb11 mutants at higher temperatures. This could be explained if dpb11 mutants activate the DNA damage checkpoint. However, shifting dpb11-1 mutant cells to the nonpermissive temperature does not result in a mobility shift of Rad53, Rad9, and Ddc1 proteins (data not shown) that normally occurs when the DNA damage checkpoint is activated. This suggests that very little DNA damage is generated in dpb11 mutants at the nonpermissive temperature. However, we cannot rule out the possibility that at intermediate temperatures in dpb11 mutants, some DNA damage is made and requires DDC1- and RAD9-dependent checkpoint signaling for survival. In contrast, in the case of HU-induced DNA replicational stress, Ddc1 plays a role distinct from that of Rad9. This role is more likely to be one of assisting repair rather than controlling cell cycle arrest.

The models we have proposed are not mutually exclusive; it is possible that they all partially reflect some in vivo situations depending on cell cycle stages and specific environments. Our data have demonstrated important physical and genetic interactions between Dpb11 and Ddc1. However, more detailed studies will be required to further understand the biochemical significance of these interactions at the molecular level.

	ACKNOWLEDGMENTS

We thank H. Ariki, M.P. Longhese, and G. Lucchini for strains and plasmids. We thank members of the Elledge lab for comments, helpful discussion, and/or reagents. This work was supported by National Institutes of Health grant GM44664 (to S.J.E.). S.J.E is an Investigator with the Howard Hughes Medical Institute and a Welch Professor of Biochemistry.

Manuscript received October 2, 2001; Accepted for publication December 10, 2001.

	LITERATURE CITED

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

ABOUSSEKHRA, A., M. BIGGERSTAFF, M. K. SHIVJI, J. A. VILPO, and V. MONCOLLIN et al., 1995  Mammalian DNA nucleotide excision repair reconstituted with purified protein components. Cell 80:859-868[Medline].

ALCASABAS, A. A., A. J. OSBORN, J. BACHANT, F. HU, and P. J. H. WERLER et al., 2001  Mrc1 transduces signals of DNA replication stress to activate Rad53. Nat. Cell Biol. 3:958-965[Medline].

ALLEN, J. B., Z. ZHOU, W. SIEDE, E. C. FRIEDBERG, and S. J. ELLEDGE, 1994  The SAD1/RAD53 protein kinase controls multiple checkpoints and DNA damage-induced transcription in yeast. Genes Dev. 8:2401-2415[Abstract/Free Full Text].

ARAKI, H., S. H. LEEM, A. PHONGDARA, and A. SUGINO, 1995  Dpb11, which interacts with DNA polymerase II(epsilon) 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[Abstract/Free Full Text].

BAI, C. and S. J. ELLEDGE, 1997  Gene identification using the yeast two-hybrid system. Methods Enzymol. 283:141-156[Medline].

BORK, P., K. HOFMANN, P. BUCHER, A. F. NEUWALD, and S. F. ALTSCHUL et al., 1997  A superfamily of conserved domains in DNA damage-responsive cell cycle checkpoint proteins. FASEB J. 11:68-76[Abstract].

BUDD, M. E. and J. L. CAMPBELL, 1995  DNA polymerases required for repair of UV-induced damage in Saccharomyces cerevisiae. Mol. Cell. Biol. 15:2173-2179[Abstract].

D'AMOURS, D. and S. P. JACKSON, 2001  The yeast Xrs2 complex functions in S phase checkpoint regulation. Genes Dev. 15:2238-2249[Abstract/Free Full Text].

DESANY, B. A., A. A. ALCASABAS, J. B. BACHANT, and S. J. ELLEDGE, 1998  Recovery from DNA replicational stress is the essential function of the S-phase checkpoint pathway. Genes Dev. 12:2956-2970[Abstract/Free Full Text].

ELLEDGE, S. J., 1996  Cell cycle checkpoints: preventing an identity crisis. Science 274:1664-1672[Abstract/Free Full Text].

GRENON, M., C. GILBERT, and N. F. LOWNDES, 2001  Checkpoint activation in response to double-strand breaks requires the Mre11/Rad50/Xrs2 complex. Nat. Cell Biol. 3:844-847[Medline].

HOLMES, A. M. and J. E. HABER, 1999  Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases. Cell 96:415-424[Medline].

HUYTON, T., P. A. BATES, X. ZHANG, M. J. STERNBERG, and P. S. FREEMONT, 2000  The BRCA1 C-terminal domain: structure and function. Mutat. Res. 460:319-332[Medline].

KAMIMURA, Y., H. MASUMOTO, A. SUGINO, and H. ARAKI, 1998  Sld2, which interacts with Dpb11 in Saccharomyces cerevisiae, is required for chromosomal DNA replication. Mol. Cell. Biol. 18:6102-6109[Abstract/Free Full Text].

KONDO, T., K. MATSUMOTO, and K. SUGIMOTO, 1999  Role of a complex containing Rad17, Mec3, and Ddc1 in the yeast DNA damage checkpoint pathway. Mol. Cell. Biol. 19:1136-1143[Abstract/Free Full Text].

KRAMATA, P., K. M. DOWNEY, and L. R. PABORSKY, 1998  Incorporation and excision of 9-(2-phosphonylmethoxyethyl)guanine (PMEG) by DNA polymerase delta and epsilon in vitro. J. Biol. Chem. 273:21966-21971[Abstract/Free Full Text].

LONGHESE, M. P., V. PACIOTTI, R. FRASCHINI, R. ZACCARINI, and P. PLEVANI et al., 1997  The novel DNA damage checkpoint protein ddc1p is phosphorylated periodically during the cell cycle and in response to DNA damage in budding yeast. EMBO J. 16:5216-5226[Medline].

LORENZ, M. C., R. S. MUIR, E. LIM, J. MCELVER, and S. C. WEBER et al., 1995  Gene disruption with PCR products in Saccharomyces cerevisiae. Gene 158:113-117[Medline].

LOWNDES, N. F. and J. R. MURGUIA, 2000  Sensing and responding to DNA damage. Curr. Opin. Genet. Dev. 10:17-25[Medline].

LYDALL, D. and T. WEINERT, 1995  Yeast checkpoint genes in DNA damage processing: implications for repair and arrest. Science 270:1488-1491[Abstract/Free Full Text].

MAKINIEMI, M., T. HILLUKKALA, J. TUUSA, K. REINI, and M. VAARA et al., 2001  BRCT domain-containing protein TopBP1 functions in DNA replication and damage response. J. Biol. Chem. 6:6.

MASUMOTO, H., A. SUGINO, and H. ARAKI, 2000  Dpb11 controls the association between DNA polymerases alpha and epsilon and the autonomously replicating sequence region of budding yeast. Mol. Cell. Biol. 20:2809-2817[Abstract/Free Full Text].

MCFARLANE, R. J., A. M. CARR, and C. PRICE, 1997  Characterisation of the Schizosaccharomyces pombe rad4/cut5 mutant phenotypes: dissection of DNA replication and G2 checkpoint control function. Mol. Gen. Genet. 255:332-340[Medline].

NAVAS, T. A., Z. ZHOU, and S. J. ELLEDGE, 1995  DNA polymerase epsilon links the DNA replication machinery to the S phase checkpoint. Cell 80:29-39[Medline].

NAVAS, T. A., Y. SANCHEZ, and S. J. ELLEDGE, 1996  RAD9 and DNA polymerase epsilon form parallel sensory branches for transducing the DNA damage checkpoint signal in Saccharomyces cerevisiae. Genes Dev. 10:2632-2643[Abstract/Free Full Text].

PACIOTTI, V., G. LUCCHINI, P. PLEVANI, and M. P. LONGHESE, 1998  Mec1p is essential for phosphorylation of the yeast DNA damage checkpoint protein ddc1p, which physically interacts with mec3p. EMBO J. 17:4199-4209[Medline].

SAKA, Y. and M. YANAGIDA, 1993  Fission yeast cut5+, required for S phase onset and M phase restraint, is identical to the radiation-damage repair gene rad4+. Cell 74:383-393[Medline].

SAKA, Y., P. FANTES, T. SUTANI, C. MCINERNY, and J. CREANOR et al., 1994a  Fission yeast cut5 links nuclear chromatin and M phase regulator in the replication checkpoint control. EMBO J. 13:5319-5329[Medline].

SAKA, Y., P. FANTES, and M. YANAGIDA, 1994b  Coupling of DNA replication and mitosis by fission yeast rad4/cut5. J. Cell Sci. 18(Suppl.):57-61.

SAKA, Y., F. ESASHI, T. MATSUSAKA, S. MOCHIDA, and M. YANAGIDA, 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[Abstract/Free Full Text].

SIKORSKI, R. S. and P. HIETER, 1989  A system of shuttle vectors and yeast host strains designed for efficient manipulation of DNA in Saccharomyces cerevisiae. Genetics 122:19-27[Abstract/Free Full Text].

SOULIER, J. and N. F. LOWNDES, 1999  The BRCT domain of the S. cerevisiae checkpoint protein Rad9 mediates a Rad9-Rad9 interaction after DNA damage. Curr. Biol. 9:551-554[Medline].

TAYLOR, R. M., B. WICKSTEAD, S. CRONIN, and K. W. CALDECOTT, 1998  Role of a BRCT domain in the interaction of DNA ligase III-alpha with the DNA repair protein XRCC1. Curr. Biol. 8:877-880[Medline].

USUI, T., H. OGAWA, and J. H. PETRINI, 2001  A DNA damage response pathway controlled by Tel1 and the Mre11 complex. Mol. Cell 7:1255-1266[Medline].

VENCLOVAS, C. and M. P. THELEN, 2000  Structure-based predictions of Rad1, Rad9, Hus1 and Rad17 participation in sliding clamp and clamp-loading complexes. Nucleic Acids Res. 28:2481-2493[Abstract/Free Full Text].

VERKADE, H. M. and M. J. O'CONNELL, 1998  Cut5 is a component of the UV-responsive DNA damage checkpoint in fission yeast. Mol. Gen. Genet. 260:426-433[Medline].

WANG, H. and S. J. ELLEDGE, 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[Abstract/Free Full Text].

WANG, Z., X. WU, and E. C. FRIEDBERG, 1993  DNA repair synthesis during base excision repair in vitro is catalyzed by DNA polymerase epsilon and is influenced by DNA polymerases alpha and delta in Saccharomyces cerevisiae. Mol. Cell. Biol. 13:1051-1058[Abstract/Free Full Text].

YAMAMOTO, R. R., J. M. AXTON, Y. YAMAMOTO, R. D. SAUNDERS, and D. M. GLOVER et al., 2000  The Drosophila mus101 gene, which links DNA repair, replication and condensation of heterochromatin in mitosis, encodes a protein with seven BRCA1 C-terminus domains. Genetics 156:711-721[Abstract/Free Full Text].

YAMANE, K. and T. TSURUO, 1999  Conserved BRCT regions of TopBP1 and of the tumor suppressor BRCA1 bind strand breaks and termini of DNA. Oncogene 18:5194-5203[Medline].

YAMANE, K., M. KAWABATA, and T. TSURUO, 1997  A DNA-topoisomerase-II-binding protein with eight repeating regions similar to DNA-repair enzymes and to a cell-cycle regulator. Eur. J. Biochem. 250:794-799[Medline].

ZHANG, X., S. MORERA, P. A. BATES, P. C. WHITEHEAD, and A. I. COFFER et al., 1998  Structure of an XRCC1 BRCT domain: a new protein-protein interaction module. EMBO J. 17:6404-6411[Medline].

This article has been cited by other articles:

				S.-h. Chen and H. Zhou Reconstitution of Rad53 Activation by Mec1 through Adaptor Protein Mrc1 J. Biol. Chem., July 10, 2009; 284(28): 18593 - 18604. [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]

				V. M. Navadgi-Patil and P. M. Burgers Yeast DNA Replication Protein Dpb11 Activates the Mec1/ATR Checkpoint Kinase J. Biol. Chem., December 19, 2008; 283(51): 35853 - 35859. [Abstract] [Full Text] [PDF]

				D. A. Mordes, E. A. Nam, and D. Cortez Dpb11 activates the Mec1-Ddc2 complex PNAS, December 2, 2008; 105(48): 18730 - 18734. [Abstract] [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]

				A. E. Burrows and S. J. Elledge How ATR turns on: TopBP1 goes on ATRIP with ATR Genes & Dev., June 1, 2008; 22(11): 1416 - 1421. [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]

				S. Delacroix, J. M. Wagner, M. Kobayashi, K.-i. Yamamoto, and L. M. Karnitz The Rad9-Hus1-Rad1 (9-1-1) clamp activates checkpoint signaling via TopBP1 Genes & Dev., June 15, 2007; 21(12): 1472 - 1477. [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]

				Y. Hashimoto, T. Tsujimura, A. Sugino, and H. Takisawa The phosphorylated C-terminal domain of Xenopus Cut5 directly mediates ATR-dependent activation of Chk1. Genes Cells, September 1, 2006; 11(9): 993 - 1007. [Abstract] [Full Text] [PDF]

				H. Ogiwara, A. Ui, F. Onoda, S. Tada, T. Enomoto, and M. Seki Dpb11, the budding yeast homolog of TopBP1, functions with the checkpoint clamp in recombination repair Nucleic Acids Res., July 13, 2006; 34(11): 3389 - 3398. [Abstract] [Full Text] [PDF]

				P. J. Lupardus and K. A. Cimprich Phosphorylation of Xenopus Rad1 and Hus1 Defines a Readout for ATR Activation That Is Independent of Claspin and the Rad9 Carboxy Terminus Mol. Biol. Cell, April 1, 2006; 17(4): 1559 - 1569. [Abstract] [Full Text] [PDF]

				A. H. Holway, C. Hung, and W. M. Michael Systematic, RNA-Interference-Mediated Identification of mus-101 Modifier Genes in Caenorhabditis elegans Genetics, March 1, 2005; 169(3): 1451 - 1460. [Abstract] [Full Text] [PDF]

				J. Jurvansuu, K. Raj, A. Stasiak, and P. Beard Viral Transport of DNA Damage That Mimics a Stalled Replication Fork J. Virol., January 1, 2005; 79(1): 569 - 580. [Abstract] [Full Text] [PDF]

				M.-G. Spiga and G. D'Urso Identification and cloning of two putative subunits of DNA polymerase epsilon in fission yeast Nucleic Acids Res., September 23, 2004; 32(16): 4945 - 4953. [Abstract] [Full Text] [PDF]

				K. Furuya, M. Poitelea, L. Guo, T. Caspari, and A. M. Carr Chk1 activation requires Rad9 S/TQ-site phosphorylation to promote association with C-terminal BRCT domains of Rad4TOPBP1 Genes & Dev., May 15, 2004; 18(10): 1154 - 1164. [Abstract] [Full Text] [PDF]

				S. M. Post, A. E. Tomkinson, and E. Y.-H. P. Lee The human checkpoint Rad protein Rad17 is chromatin-associated throughout the cell cycle, localizes to DNA replication sites, and interacts with DNA polymerase {epsilon} Nucleic Acids Res., October 1, 2003; 31(19): 5568 - 5575. [Abstract] [Full Text] [PDF]

				S. Harris, C. Kemplen, T. Caspari, C. Chan, H. D. Lindsay, M. Poitelea, A. M. Carr, and C. Price Delineating the position of rad4+/cut5+ within the DNA-structure checkpoint pathways in Schizosaccharomyces pombe J. Cell Sci., September 1, 2003; 116(17): 3519 - 3529. [Abstract] [Full Text] [PDF]

				A. J. Osborn and S. J. Elledge Mrc1 is a replication fork component whose phosphorylation in response to DNA replication stress activates Rad53 Genes & Dev., July 15, 2003; 17(14): 1755 - 1767. [Abstract] [Full Text] [PDF]

				R. P. St.Onge, B. D. A. Besley, J. L. Pelley, and S. Davey A Role for the Phosphorylation of hRad9 in Checkpoint Signaling J. Biol. Chem., July 11, 2003; 278(29): 26620 - 26628. [Abstract] [Full Text] [PDF]

				M. Chang, M. Bellaoui, C. Boone, and G. W. Brown A genome-wide screen for methyl methanesulfonate-sensitive mutants reveals genes required for S phase progression in the presence of DNA damage PNAS, December 24, 2002; 99(26): 16934 - 16939. [Abstract] [Full Text] [PDF]

				R. A. Van Hatten, A. V. Tutter, A. H. Holway, A. M. Khederian, J. C. Walter, and W. M. Michael The Xenopus Xmus101 protein is required for the recruitment of Cdc45 to origins of DNA replication J. Cell Biol., November 25, 2002; 159(4): 541 - 547. [Abstract] [Full Text] [PDF]