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Vol. 17, Issue 8, 3456-3468, August 2006

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Rad4^TopBP1, a Scaffold Protein, Plays Separate Roles in DNA Damage and Replication Checkpoints and DNA Replication

Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305-5324

Submitted January 23, 2006; Revised May 12, 2006; Accepted May 15, 2006
Monitoring Editor: Mark Solomon

	ABSTRACT

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Rad4^TopBP1, a BRCT domain protein, is required for both DNA replication and checkpoint responses. Little is known about how the multiple roles of Rad4^TopBP1 are coordinated in maintaining genome integrity. We show here that Rad4^TopBP1 of fission yeast physically interacts with the checkpoint sensor proteins, the replicative DNA polymerases, and a WD-repeat protein, Crb3. We identified four novel mutants to investigate how Rad4^TopBP1 could have multiple roles in maintaining genomic integrity. A novel mutation in the third BRCT domain of rad4^+TopBP1 abolishes DNA damage checkpoint response, but not DNA replication, replication checkpoint, and cell cycle progression. This mutant protein is able to associate with all three replicative polymerases and checkpoint proteins Rad3^ATR-Rad26^ATRIP, Hus1, Rad9, and Rad17 but has a compromised association with Crb3. Furthermore, the damaged-induced Rad9 phosphorylation is significantly reduced in this rad4^TopBP1 mutant. Genetic and biochemical analyses suggest that Crb3 has a role in the maintenance of DNA damage checkpoint and influences the Rad4^TopBP1 damage checkpoint function. Taken together, our data suggest that Rad4^TopBP1 provides a scaffold to a large complex containing checkpoint and replication proteins thereby separately enforcing checkpoint responses to DNA damage and replication perturbations during the cell cycle.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Eukaryotic cells have evolved a complex network of genomic surveillance mechanisms "checkpoints" to maintain genomic integrity in the face of various genomic insults during cell cycle progression. Checkpoint responses detect the genomic perturbations by sensor proteins, which then relay the signal to transducer proteins, and to effectors to transiently arrest the cell cycle (Nyberg et al., 2002). Failure to enforce the correct checkpoint responses can result in the accumulation of mutations and chromosomal rearrangements, which are the hallmarks of cancer cells (Bakkenist and Kastan, 2004; Kastan and Bartek, 2004; Lukas and Bartek, 2004).

Rad4/Cut5 of fission yeast and its orthologues, budding yeast DPB11, Drosophila Mus101, and mammalian TopBP1, are essential for both DNA replication and checkpoint responses, giving them a unique role in genome maintenance (Saka and Yanagida, 1993; Saka et al., 1994a, 1994b; Araki et al., 1995; McFarlane et al., 1997; Verkade and O’Connell, 1998; Makiniemi et al., 2001; Harris et al., 2003; Furuya et al., 2004; Garcia et al., 2005). Rad4/Cut5 (hereafter termed Rad4^TopBP1) in mouse and fission yeast is also involved in monitoring meiotic checkpoint (Perera et al., 2004). Rad4^TopBP1 of fission yeast contains four BRCT domains has been show to interact with a BRCT-repeat protein, Crb2, an adaptor for Chk1 activation, as well as a WD-repeat protein, Crb3, by two-hybrid criteria (Saka et al., 1997; Garcia et al., 2005). The fission yeast Crb3 is an ortholog of mammalian Wdr18 (Killian and Hubbard, 2004; NCBI, database). It is essential for viability of fission yeast although its physiological role is unknown (Saka et al., 1997).

It is intriguing that Rad4^TopBP1, a single protein, is required for both DNA replication and checkpoint responses in genome maintenance. Despite numerous intense studies, the mechanism by which Rad4^TopBP1 coordinates its multiple roles in maintaining genome integrity is unclear. Studies of Rad4^TopBP1 of fission yeast have shown that phosphorylation of the checkpoint clamp component, Rad9, on Thr⁴¹² and Ser⁴²³ in response to damage promotes Rad9 protein to associate with two C-terminal BRCT domains of Rad4^TopBP1. This association is a prerequisite for activation of the Chk1 damage checkpoint but not the Cds1 replication checkpoint. Furthermore, Rad4^TopBP1 is able to coprecipitate with Rad3^ATR when Rad9 is phosphorylated at Thr⁴¹² and Ser⁴²³. The study suggests that phosphorylation of Rad9 at Thr⁴¹² and Ser⁴²³ coordinates the formation of an active checkpoint complex, which depends on the physical involvement of Rad4^TopBP1 (Furuya et al., 2004). Moreover, a recent study of Rad4^TopBP1 of both Xenopus and human has shown that Rad4^TopBP1 plays a critical role in the initiation of ATR-dependent checkpoint signaling processes (Kumagai et al., 2006).

We show here that Rad4^TopBP physically associates with the checkpoint sensor proteins and the replicative DNA polymerases. We identified four novel mutants of rad4^+TopBP1 to investigate how Rad4^TopBP1 coordinates its multiple roles to maintain genomic integrity. A detailed analysis of one mutant having a mutation in the third BRCT motif (R3) indicates that the role of Rad4^TopBP in checkpoint responses to DNA damage can be separated from the checkpoint response to replication perturbation and from its role in DNA replication and cell cycle progression. Furthermore, genetic and biochemical analyses suggest that Crb3 transiently associates with Rad4^TopBP1; Crb3 has a role in maintaining the DNA damage checkpoint and seems to influence the DNA damage checkpoint function of Rad4^TopBP1. Taken together, our results suggest that Rad4^TopBP1 functions as a scaffold in a large protein complex containing both checkpoint proteins and replication proteins to selectively enforce checkpoint response to DNA damage or replication perturbation, or DNA replication in order to maintain genomic integrity during the cell cycle.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Strains and Media
Schizosaccharomyces pombe strains were grown in YES or EMM medium containing nutritional supplements as necessary. Standard genetic methods, molecular biological techniques, and generation of tagged strains were as described in Moreno et al. (1991) and Bahler et al. (1998). GFP(S65T) tag was constructed to the C-terminus of rad4^+TopBP1 and rad4-c11^TopBP1 at its genomic locus, and GFP-tagged Rad4^TopBP1 and Rad4-c11^TopBP1 are nuclear proteins (unpublished data). Cells containing GFP epitope–tagged rad4^+TopBP1 (rad4^+TopBP1:GFP) and rad4-c11^TopBP1 (rad4-c11^TopBP1:GFP) have phenotype and properties identical to that of the wild-type and rad4-c11^TopBP1 cells, respectively (Supplementary Figure 1). Diploid strain crb3/+ with one copy of the crb3⁺ deleted was generated using PCR-mediated gene disruption (Bahler et al., 1998). Other diploid strains were generated by protoplast fusions or by crossing two homothallic diploid strains with complementing ade6 markers. The ade⁺ diploids are selected and analyzed by PCR and confirmed by sequencing. All strains used in this study are listed in Supplementary Tables 1 and 2.

View larger version (45K): [in this window] [in a new window]   Figure 1. Characterization of novel rad4TopBP1 mutants. (A) Schematic locations of the novel rad4TopBP1 mutations. Rad4TopBP1 consists of four BRCT domains (R1, R2, R3, and R4) and two hydrophilic (acidic and basic) domains. Locations of the novel mutants and the previously characterized mutant rad4-116TopBP1 in each BRCT domain are marked. (B) Sensitivities of the rad4TopBP1 mutants to genotoxic agents. Cells were cultured to log phase, and then 10-fold serial dilutions of 1 x 107 cells were spotted onto YES plates or YES plates with 5 mM HU, 11 mM caffeine, 10 µM CPT, or 0.005% MMS and incubated at 30°C for 3 d. (C) UV sensitivity of rad4TopBP1 mutants. Wild-type and rad4TopBP1 mutants were grown in YES to early log phase, and then >1000 cells were plated in triplicate on YES plates. Cells were irradiated with the indicated doses of UV and incubated at 25°C for 5 d. Data shown represents the average results of three independent experiments.

Preparation of Protein Extract and Immunoprecipitation and Immunoblotting of Rad4^TopBP1:GFP
Cells were harvested by centrifugation and washed once with ice-cold stop buffer (150 mM NaCl, 50 mM NaF, 10 mM EDTA, 1 mM NaN₃, pH 8.0) and once with LT500 lysis buffer (25 mM Tris-HCl, pH 7.4, 500 mM NaCl, 1 mM EDTA, 10% glycerol, 10 mM -mercapthoethanol). The cells were resuspended in LT500 lysis buffer supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF) and 2x protease inhibitors (Roche Molecular Biochemicals, Indianapolis, IN) and frozen as pellets by dropping the cell suspension into liquid nitrogen. The frozen cell pellets were broken in the dry ice powder using a coffee grinder. The homogenate was resuspended in LT300 lysis buffer (25 mM Tris-HCl, pH 7.4, 300 mM NaCl, 1 mM EDTA, 10% glycerol, 0.1% Nonidet P-40, 10 mM -mercapthoethanol, and 1 mM PMSF, supplemented with complete protease inhibitors; Roche Molecular Biochemicals), vortexed, sonicated, treated with DNAse I (Invitrogen, Carlsbad, CA), and then centrifuged to prepare a cleared whole-cell extract. Protein concentration was determined using the Bio-Rad protein assay (Richmond, CA).

Three milligrams of total protein was diluted to 300 µl with LT300 lysis buffer and incubated with mouse anti-GFP monoclonal antibody (Roche Molecular Biochemicals) at 4°C for 2 h. Protein G plus protein A-Agarose beads (50 µl, Oncogene Research Products, Boston, MA) were added and incubated at 4°C for 1 h. Immunoprecipitates were washed four times with LT300 lysis buffer and resuspended in 30 µl of 2x SDS loading buffer. For immunoblot analysis, 40–60 µg of total protein was loaded for detection of protein in input lysates. Ten micrograms of total immunoprecipitated materials was used in SDS-PAGE for detection of immunoprecipitation, and 20 µg was used for detection of the coimmunoprecipitation. Extracts were separated on SDS-PAGE and transferred to PVDF membrane (Bio-Rad). Two protein bands marked with asterisks were Rad4^TopBP1:GFP in full-length (100 kDa) and in N-terminal truncated (93-kDa) in all immunoprecipitates. Immunoblots were probed with appropriate antibodies: mouse anti-GFP antibody (1:1000; Roche Molecular Biochemicals) for Rad4^TopBP1; mouse anti-M2 FLAG antibody (1:1000; Sigma) for FLAG-tagged polymerases and ; mouse anti-myc (9E10; 1:2000) for myc-tagged Rad3^ATR, Hus1, and Rad17; and chicken B18 anti-pol (1:1000) for polymerase (Park et al., 1993). Immunoreactive bands were revealed with HRP-conjugated secondary goat anti-mouse, anti-rabbit IgG antibody (1:10,000), or anti-chicken IgG antibody (1:5000; New England BioLabs, Beverly, MA) and the luminol-based ECL detection kit (Perkin Elmer-Cetus, Norwalk, CT). Immunoprecipitation of Rad26^ATRIP was performed by rabbit anti-Rad26^ATRIP (1:1000) and probed the immunoblot by anti-GFP (1:1000) for Rad4^TopBP1:GFP or Rad4-c11^TopBP1:GFP.

Cds1 Immunoprecipitation and Kinase Assay
Immunoprecipitation of Cds1 protein and Cds1 kinase activity were performed as described (Lindsay et al., 1998).

Chk1 Immunoprecipitation
Protein extraction was performed with glass beads in LT300 lysis buffer using FastPrep (BIO 101, Carlsbad, CA) vortexing machine. For Chk1 immunoprecipitation (IP) in the rad4^+TopBP1:GFP cells and mutant rad4-c11^TopBP1:GFP cells, 5 mg of soluble protein was added to 100 µl of anti-HA (3F10 [PDB] ) affinity matrix (Roche Molecular Biochemicals) and incubated at 4°C with rocking for 5 h. Immunoprecipitates were washed four times with LT300 lysis buffer and resuspended in 2x SDS gel loading buffer. Ten microliters of each IP were separated on 8% SDS-PAGE, transferred to PVDF membrane (Bio-Rad), and detected by anti-HA 12CA5 (1:500; Roche Molecular Biochemicals).

DAPI Staining
Cells were collected and fixed in 100% methanol at –20°C for at least 20 min. Cells were then washed three times in 1x PEM buffer (Sawin and Nurse, 1998), resuspended in 20–50 µl 1x PEM, and stained with 1 µg/ml DAPI. All images were photographed with a Nikon epifluorescence microscope (Melville, NY).

Two-Hybrid Assay
The full-length crb3⁺ and rad9⁺ was fused to the 3' end of the LexA DNA-binding domain in pEG202 (Gyuris et al., 1993) to generate a BamHI/NotI crb3⁺ and rad9⁺ fragment by PCR and to clone into the BamHI/NotI sites to yield the bait plasmids. The wild-type R3 and mutant R3-c11 domain of rad4^TopBP1 was fused to LexA DNA-binding domain in pEG202 by first generating a BamHI/NotI R3 and R3-c11 rad4^TopBP1 fragment by PCR and then cloning into the BamHI/NotI sites, to yield the bait plasmid. The full-length rad4^+TopBP1, cds1⁺, chk1⁺, and crb3⁺ were independently constructed to the 3'end of the GAL4 transcriptional activation domain and cloned into the EcoRI/XhoI sites. rad4^TopBP1 fragments deleted for the first two BRCT domains (R1 and R2) region, R1R2rad4^TopBP1 and R1R2rad4-c11^TopBP1, which contains a mutation in the R3 region, were independently fused to the 3' end of the GAL4 transcriptional activation domain and cloned into the EcoRI/XhoI sites. The two-hybrid experiments were performed with S. cerevisiae strain Y1003. Activities of -galactosidase were expressed in Miller units as the mean of six independent determinations (±SD).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Characterization of rad4^TopBP1 Mutants
We devised a genetic screen using cdc20-M10 (a pol mutant) to identify genetic elements that are involved in activating Chk1 after early S-phase arrest (Griffiths et al., 2000). Four novel rad4^+TopBP1 mutants were isolated by this screen among many of the checkpoint mutants. All four novel rad4^TopBP1 mutants reside in the BRCT domains of Rad4^TopBP1 (Figure 1A). One mutant, rad4-c23^TopBP1, contains an His⁴⁶ to Tyr (H46Y) substitution residing in the first BRCT domain (R1). This mutation is immediately adjacent to the previously identified, and well-documented, thermosensitive rad4-116^TopBP1 mutant, which contains a Thr⁴⁵ to Met (T45M) substitution and is defective in checkpoint responses to damage and replication perturbations at its restrictive temperature (Saka et al., 1994a, 1997; Harris et al., 2003). Interestingly, rad4-c23^TopBP1 is not thermosensitive (Figure 1B). Two new mutants, rad4-m15^TopBP1 and rad4-c17^TopBP1, contain A155T and S171N substitutions, respectively, within the second BRCT domain (R2). rad4-c17^TopBP1 is the only temperature-sensitive mutant isolated (Figure 1, A and B). One mutant, rad4-c11^TopBP, contains an E368K substitution within the third BRCT domain (R3; Figure 1A). The rad4-c11^TopBP1 mutation is localized within the conserved motif (consensus W-X-X-X-C/S) found in all BRCT domains.

These novel rad4^TopBP1 mutants were tested for their sensitivity to different genotoxic agents. The rad4^TopBP1 mutants exhibit varying levels of sensitivity to UV with rad4-c11^TopBP1 exhibiting a slightly higher UV sensitivity than rad4-116^TopBP1 (Figure 1C; Saka and Yanagida, 1993; McFarlane et al., 1997). rad4-c11^TopBP1 and rad4-116^TopBP1 exhibit similar sensitivity to hydroxyurea (HU), caffeine (caf), CPT, and methyl methanesulfonate (MMS) at 30°C (Figure 1B). In contrast to rad4-116^TopBP1, mutant rad4-c23^TopBP1, containing a mutation immediately adjacent to rad4-116^TopBP1, is only sensitive to caffeine and mildly sensitive to UV (Figure 1, B and C). rad4-116^TopBP1, rad4-m15^TopBP1, and rad4-c11^TopBP1 are all sensitive to -radiation, whereas rad4-c23^TopBP1 and rad4-c17^TopBP1 are not -radiation sensitive (unpublished data). These results indicate that mutations in different alleles, even adjacent alleles in the same BRCT-domain, can induce disparate phenotypes in cells.

Mutation in rad4-c11^TopBP1 Abolishes Cells' Chk1 Checkpoint Responses to DNA Damage But Not the Cds1 Response to Replication Perturbation
Rad4^TopBP1 is required to enforce checkpoint responses to replication stress and DNA damage (Garcia et al., 2005). We thus analyzed whether the new rad4^TopBP1 mutants could compromise replication checkpoint by analyzing the Cds1 kinase activation in response to HU treatment. The Cds1 kinase activity in the HU-treated thermosensitive mutants rad4-116^TopBP1 and rad4-c17^TopBP1 was activated to a comparable extent as in wild-type cells at the permissive temperature of 26°C and at the semipermissive temperature of 32°C (Figure 2A). As expected, Cds1 kinase activity was dramatically reduced in rad4-116^TopBP1 and reduced to a lesser extent in rad4-c17^TopBP1 at the restrictive temperature of 36°C (Figure 2A). The non–temperature-sensitive mutants rad4-c23^TopBP1, rad4-m15^TopBP1, and rad4-c11^TopBP1 are all proficient in Cds1 kinase activation (Figure 2B).

View larger version (51K): [in this window] [in a new window]   Figure 2. Activation of Cds1 and Chk1 kinases in rad4TopBP1 mutants. Cds1 kinase assays were performed as described in Material and Methods. Chk1 activation was measured by the phosphorylation-dependent mobility shift of Chk1 protein in rad4+TopBP1:GFP and rad4TopBP1:GFP mutants cells containing an integrated HA-tagged chk1+ as described in Materials and Methods. (A) Cds1 kinase activity of the thermosensitive mutants. (B) Cds1 kinase activity of the nonthermosensitive mutants. (C) Chk1 phosphorylation of the thermosensitive mutants, rad4-116TopBP1 and rad4-c17TopBP1 and nonthermosensitive mutants rad4-m15TopBP1 and rad4-c23TopBP1. (D) Mutation in rad4-c11TopBP1 abolishes Chk1 activation. Mutant rad4-c11TopBP1 cells were treated with CPT for 2 h (top panel) and 4 h and 6 h (bottom panel). (E) rad4-c11TopBP1 mutant is defective in delay of mitotic entry in response to damage. Cells were synchronized by cdc25-22 block and release and then treated with or without 30 µM of CPT. The synchronous cdc25-22 rad4+TopBP1 (top panel), cdc25-22 chk1 (middle panel), and cdc25-22 rad4-c11TopBP1 (bottom panel) cells were scored for septation index at 30-min intervals. Solid symbols represent cells not treated with CPT; open symbols are cells treated CPT. (F) Deletion of chk1+ in rad4-c11TopBP1 has a synergistic effect on the cells' sensitivity to chronic CPT treatment. Cells were cultured to log phase and then 10-fold serial dilutions of 1 x 107 cells were spotted onto YES plates or YES plates with 2.5 µM CPT and incubated at 30°C for 3 d.

To ensure that mutation in rad4-c11^TopBP1 compromises the Chk1-mediated G2/M phase checkpoint, we analyzed the kinetics of mitotic entry of rad4-c11^TopBP1 cells. Mutant rad4-c11^TopBP1 was constructed in cdc25-22 background and synchronized by cdc25-22 block at G2 and release. The percent of cells entering mitosis upon CPT treatment was measured by septation index and compared with that of cdc25-22 cells with wild-type rad4^+TopBP1 and double mutant chk1 cdc25-22. As expected, cells with mutation in the rad4-c11^TopBP1 or deletion of chk1⁺ did not delay the mitotic entry in response to CPT treatment, whereas cells with rad4^+TopBP1 did not enter mitosis after CPT treatment (Figure 2E). We further tested the effect of deletion of chk1⁺ in rad4-c11^TopBP1 after chronic exposure to CPT on solid media. After chronic exposure to 2.5 µM CPT, the two single mutants exhibited similar viability. Interestingly, deletion of chk1⁺ in rad4-c11^TopBP1 had a synergistic effect on the double mutant's viability (Figure 2F). This result suggests that chronic exposure of rad4-c11^TopBP1 to CPT may compromise another damage response pathway in addition to the response that activates Chk1 upon acute CPT treatment (Figure 2, A–E). These results support the notion that cellular responses to acute and chronic DNA damage are different and underscore the importance of the multiple roles of Rad4^TopBP1 in genome maintenance.

Chk1 is not only activated in response to replication fork collapse induced by CPT (Figure 3A) but also by HU in the absence of Cds1 (Figure 3B; Boddy et al., 1998; Lindsay et al., 1998). To further determine whether the rad4-c11^TopBP1 mutant is also defective in Chk1 activation in response to DNA damage caused by HU-induced replication fork stalling in the absence of Cds1, we created a mutant strain cds1 rad4-c11^TopBP1chk1:HA to assess the phosphorylation of Chk1 protein in response to HU treatment. On HU treatment, Chk1 was activated in the cds1 chk1:HA strain (Figure 3B). In contrast, no Chk1 phosphorylation was observed in the cds1 rad4-c11^TopBP1 chk1:HA strain after HU treatment (Figure 3B). Furthermore, upon HU treatment, only the cds1 rad4-c11^TopBP1 double mutant, deficient in both cds1⁺ and chk1⁺ activation, exhibited a reduced level of Cdc2 phosphorylation (Figure 3C). These results indicate that in the cds1 rad4-c11^TopBP1 double mutant, the Chk1-mediated mitotic checkpoint in response to HU treatment is abolished. Thus, rad4-c11^TopBP1 mutant has a defective Chk1 activation in response to HU-treated cds1 cells.

View larger version (44K): [in this window] [in a new window]   Figure 3. Mutations in rad4-c11TopBP1 abolish Chk1 activation in cds1 cells. (A) The Chk1 phosphorylation was assayed in chk1:HA and cds1 chk1:HA mutant cells that were incubated with 30 µM CPT for 2 h at 30°C. (B) Cells with mutation in rad4-c11TopBP1 fail to activate Chk1 in the absence of cds1+. chk1:HA, cds1 chk1:HA, and cds1 chk1:HA rad4-c11TopBP1 strains were incubated with 12 mM HU for 4 h at 30°C. Cell extracts were examined for phosphorylation dependent mobility shift of Chk1 in response to HU-induced damage in the absence of functional Cds1. (C) Phosphorylation of Cdc2 in HU-treated cds1 rad4-c11TopBP1 double mutant is compromised. Wild-type rad4+TopBP1 and mutants, rad4-c11TopBP1, cds1, and cds1 rad4-c11TopBP1 cells were grown in YES media in the presence of 12 mM HU for the 4 h at 30°C and examined for Cdc2 phosphorylation levels. Cdc2 (PSTAIRE) was used as a loading control. (D) Deletion of cds1+ in rad4-c11TopBP1 induces mitotic catastrophic phenotype in response to HU-induced damage. Wild-type, rad4-c11TopBP1, cds1, and cds1 rad4-c11TopBP1 cells were grown in YES media in the presence of 12 mM HU for the indicated times at 30°C, fixed, and then stained with DAPI to determine % cut phenotype. Percentage cut phenotype represents the average result of two independent experiments of counting 300 cells. Arrows indicate cells with abnormal nucleus.

Rad4^TopBP1 Protein Associates with Checkpoint Sensor Proteins and Mutation in rad4-c11^TopBP1 Reduces the Mutant's Rad9 Phosphorylation
Chk1 activation in response to DNA damage depends on the checkpoint complexes, Rad3^ATR-Rad26^ATRIP, Rad9-Rad1-Hus1, Rad17-Rfc_2-5, and Rad4^TopBP1 (O’Connell and Cimprich, 2005). A recent study has shown that association of Rad4^TopBP1 with a phosphorylated form of Rad9 is required to promote Chk1 activation (Furuya et al., 2004). Given that the mutation in rad4-c11^TopBP1 compromises Chk1 phosphorylation in response to CPT-induced damage (Figures 2 and 3), we examined the physical association of Rad4^TopBP1 or Rad4-c11^TopBP1 protein with all of the checkpoint sensor proteins, with or without CPT treatment. Wild-type and mutant Rad4^TopBP1:GFP protein were detected in the anti-Rad26^ATRIP immunoprecipitates from extracts of rad4^+TopBP1:GFP and rad4-c11^TopBP1:GFP cells (Figure 4A). Rad3^ATR:myc was detected in the anti-GFP immunoprecipitates from extracts of rad4^+TopBP1:GFP rad3^+ATR:myc and rad4-c11^TopBP1:GFP rad3^+ATR:myc cells after either CPT or HU treatment (Figure 4B). In contrast to that reported in Furuya et al. (2004), no significant increased level of Rad3^ATR associated with either Rad4^TopBP1 or Rad4-c11^TopBP1 was detected after treatment with these genotoxic agents (Figure 4B). The discrepancy may due to the damaged structures induced by these genotoxic agents being different from those induced by -radiation used by Furuya et al. Furthermore, Hus1:myc and Rad17:myc were detected in the GFP immunoprecipitates from cell extracts of rad4^+TopBP1:GFP hus1⁺:myc, rad4-c11^TopBP1:GFP hus1⁺:myc (Figure 4C), and rad4^+TopBP1:GFP rad17⁺:myc and rad4-c11^TopBP1:GFP rad17⁺:myc cells (Figure 4D). These results indicate that Rad4^TopBP1 associates with Rad3^ATR-Rad26^ATRIP, Hus1, and the clamp loader Rad17 in vivo, independent of damage, thus suggesting that Rad4^TopBP1 physically coexists with these checkpoint sensor proteins in a complex. Importantly, the mutation in rad4-c11^TopBP1 does not affect the association of mutant Rad4-c11^TopBP1 protein with Rad3^ATR -Rad26^ATRIP, Hus1, Rad9, and Rad17 checkpoint sensor proteins. Hence, failure to activate the Chk1 damage checkpoint in rad4-c11^TopBP1 is not due to the inability of Rad4-c11^TopBP1 to associate with checkpoint sensor proteins.

View larger version (41K): [in this window] [in a new window]   Figure 4. Rad4TopBP1 physically associates with checkpoint sensor proteins. (A) Coprecipitation of Rad4TopBP1:GFP or Rad4-c11TopBP1:GFP proteins with Rad26ATRIP. (B) Coprecipitation of Rad4TopBP1:GFP or Rad4-c11TopBP1:GFP with Rad3ATR:myc from extracts of rad4+TopBP1:GFP rad3+ATR:myc cell or rad4-c11TopBP1:GFP rad3+ATR:myc cells treated with CPT or HU. (C) Coprecipitation of Hus1:myc with Rad4TopBP1: GFP or Rad4-c11TopBP1:GFP from extracts of rad4+TopBP1:GFP hus1+:myc or rad4-c11TopBP1:GFP hus1+:myc cells. (D) Coprecipitation of Rad17:myc with Rad4TopBP1:GFP or Rad4-c11TopBP1:GFP from extracts of rad4+TopBP1:GFP rad17+:myc or rad4-c11TopBP1:GFP rad17+:myc cells. Cell cultures were grown in YES media in the absence (–CPT) or presence (+CPT) of 30 µM CPT for 2 h at 30°C. Immunoprecipitations were performed as described in Materials and Methods. (E) Rad9 interacts with Rad4TopBP1 and Rad4-c11TopBP1 by two-hybrid assay. (F) Rad4TopBP1 and Rad4-c11TopBP1 physically associate with Rad9 in vivo. Rad9:HA was immunoprecipitated by anti-HA (3F10) affinity matrix from extracts of rad9:HA rad4+TopBP1:GFP or rad9:HA rad4-c11TopBP1:GFP cells. The Rad9 immunoprecipitates were then probed by immunoblotting with mouse anti-HA (12CA5). Coprecipitation of Rad9:HA with Rad4TopBP1:GFP or Rad4-c11TopBP1:GFP was detected by rabbit anti-GFP (Abcam, Cambridge, United Kingdom). The two arrows marked with asterisks in Rad4TopBP1:GFP immunoprecipitation denote full-length and N-terminal truncation of the Rad4TopBP1 protein. IgG was used as control of the immunoprecipitation. -tubulin or Cdc2 (PSTAIRE) were used as input loading control.

To confirm this observed interaction in vivo, we examined coimmunoprecipitation of Rad9 with Rad4^TopBP1. In either asynchronous cells or CPT-treated cells, Rad4^TopBP1 and Rad4-c11^TopBP1 proteins were detected in the anti-HA-Rad9 immunoprecipitates, thus supporting the two-hybrid finding that mutation in rad4-c11^TopBP1 does not abolish the association of Rad4-c11^TopBP1 mutant protein with Rad9 (Figure 4F, lanes 1–4). Furuya et al. (2004) have also shown that Rad9 phosphorylated at Thr²²⁵ (T225) results in a slow mobility form of Rad9 protein in gel and that the Rad9-T225 phosphorylation is dependent on the prior phosphorylation at Rad9-T412/S423. Upon CPT treatment, the Rad9-T225 phosphorylation was substantially reduced in rad4-c11^TopBP1 (Figure 4F, compare lane 2 with lane 4). These results support the notion proposed by Furuya et al. that phosphorylations of Rad9 at T412/S423 promote the interaction of Rad4^TopBP1 with Rad9 and Rad3^ATR and the interaction is required to activate Chk1 damage checkpoint.

The Mutation in rad4-c11^TopBP1 Does Not Affect DNA Replication or Cell Cycle Progression
The association of budding yeast Pol1^Pol and Pol2^Pol to the origin of DNA replication is thought to require Dpb11^TopBP1, and this association functions mainly during initiation of DNA replication (Araki et al., 1995; Masumoto et al., 2000). However, human TopBP1 has been shown to associate with Pol throughout the cell cycle (Makiniemi et al., 2001). Finding that the rad4-c11^TopBP1 mutation compromises the cells' Chk1 damage checkpoint response prompted us to test whether this mutation had effects on the replication function of Rad4^TopBP1 and cell cycle progression.

We first examined the physical association of Rad4^TopBP1 and mutant Rad4-c11^TopBP1 proteins with the replicative DNA polymerases, Pol, Pol, and Pol. Immunoprecipitates of anti-GFP of GFP-tagged Rad4^TopBP1 and Rad4-c11^TopBP1 proteins from extracts of rad4^+TopBP1:GFP or rad4-c11^TopBP1:GFP cells harboring either pol⁺:Flag or pol⁺:Flag were probed for coprecipitation of Pol or Pol using an anti-Flag antibody (Figure 5A). Flag-tagged Pol and Pol proteins were detected in the GFP immunoprecipitates from extract of asynchronous cells and HU- or CPT-treated cells expressing the GFP-tagged Rad4^TopBP1 or Rad4-c11^TopBP1 (Figure 5A). Similarly, GFP-tagged Rad4^TopBP1 or Rad4-c11^TopBP1 was detected in Pol-immunoprecipitates from cell extracts of either rad4^+TopBP1:GFP or rad4-c11^TopBP1:GFP (Figure 5B). These results indicate that Rad4^TopBP1 associates with all three replicative polymerases in a protein complex not only during initiation of DNA replication but also in predominantly G2 phase of the fission yeast cell cycle. Importantly, the mutation in rad4-c11^TopBP1 does not affect the Rad4-c11^TopBP1 protein's ability to associate with the replicative polymerases in the complex. Thus, failure to activate Chk1 damage checkpoint in rad4-c11^TopBP1 is not due to the inability of Rad4-c11^TopBP1 protein to associate with the replicative polymerases.

View larger version (47K): [in this window] [in a new window]   Figure 5. Association of Rad4TopBP1 and replicative DNA polymerases and mutation in rad4-c11TopBP1 does not affect the association and cell cycle progression. (A) Coprecipitation of Rad4TopBP1:GFP or Rad4-c11TopBP1:GFP with Pol and Pol. Cells with pol+:Flag or pol+:Flag in rad4+TopBP1:GFP or rad4-c11TopBP1:GFP were grown in YES media and treated with either 30 µM CPT for 2 h or 12 mM HU for 4 h at 30°C. Cell extracts were immunoprecipitated with anti-GFP antibody and probed with anti-Flag antibody for coprecipitation of Pol:Flag or Pol:Flag. (B) Coprecipitation of Rad4TopBP1:GFP or Rad4-c11TopBP1:GFP with Pol. Cell extracts of rad4+TopBP1:GFP or rad4-c11TopBP1:GFP were immunoprecipitated with anti-Pol antibody cross-linked onto Agarose beads, and the immunoprecipitates were probed with anti-GFP. The coprecipitations of the replicative DNA polymerases with Rad4TopBP1or Rad4-c11TopBP1 were not caused by the presence of cellular DNA as shown in the ethidium bromide (EtBr) control. The two arrows marked with asterisks in Rad4TopBP1:GFP immunoprecipitation denote full-length and N-terminal truncation of the Rad4TopBP1 protein. IgG was used as control of immunoprecipitation. -tubulin or Cdc2 (PSTAIRE) were used as input loading control. (C) rad4-c11TopBP1 mutant has normal cell cycle progression. rad4+TopBP1:GFP cdc25-22 and rad4-c11TopBP1:GFP cdc25-22 cells were arrested in G2 at 36°C and released to mitosis by shift to 26°C. Cell extracts were prepared in every 20 min and analyzed for Cdc2 phosphorylation by immunoblotting with polyclonal antibodies to phospho-Cdc2 (Tyr15) and Cdc2 (PSTAIRE). rad4+TopBP1:GFP cdc25-22 () and rad4-c11TopBP1:GFP cdc25-22 () cells were examined microscopically for the septation index (SI), and both wild-type and mutant cells exhibited identical septation index.

Rad4-c11^TopBP1 Protein Has Compromised Interaction with Crb3
We further explored what other protein–protein interactions that could be compromised by the rad4-c11^TopBP1 mutation, which might explain the failure to activate the Chk1 damage checkpoint. Two-hybrid analysis using either the R1+R2 or the R3 domain of Rad4^TopBP1 has previously identified the interactions with Crb2 and Crb3, respectively (Saka et al., 1997). Fission yeast crb3⁺ is an essential gene, and its gene product is thought to be involved in the G1/S progression (Saka et al., 1997). However, the biological significance of the interaction between the R3 domain of Rad4^TopBP1 and Crb3 is unknown.

Given that the mutation in rad4-c11^TopBP1 is in the R3 domain, we used two-hybrid analysis to examine whether this mutation had any adverse effects on the interaction of Rad4^TopBP1 with Crb3. Both Crb3 and Chk1 interact with the R3 domain of Rad4^TopBP1 as bait (Figure 6A). In striking contrast, both Crb3 and Chk1 failed to interact with the R3 domain of Rad4-c11^TopBP1. We also performed this analysis using crb3⁺ as bait and full-length rad4^+TopBP1 or rad4^TopBP1 containing a deletion of R1 and R2 domains (R1R2rad4 in Figure 6B) as targets. Both full-length and R1R2rad4 target constructs were proficient in interacting with Crb3. Again, the interaction between Crb3 and Rad4-c11^TopBP1 with deletion of R1 and R2 motifs (R1R2rad4-c11 in Figure 6B) was reduced to the same level as Crb3 by itself, which exhibited a low level of transactivation. Thus, the two-hybrid results confirm the previous report (Saka et al., 1997) that the R3 domain of Rad4^TopBP1 interacts with Crb3. Importantly, mutation in the R3 domain of rad4-c11^TopBP1 significantly reduces the extent of interaction of Rad4-c11^TopBP1 with Crb3 and Chk1 (Figure 6A).

View larger version (18K): [in this window] [in a new window]   Figure 6. Two-hybrid assay of the interaction of wild-type Rad4TopBP1 or mutant Rad4-c11TopBP1 with Crb3. (A) R3 domain of Rad4TopBP1 interacted with Crb3 and Chk1 and Rad4-c11TopBP1 R3 domain failed to interact with both Crb3 and Chk1. Two-hybrid assay with Rad4TopBP1 R3 BRCT or Rad4-c11TopBP1 R3 BRCT domain as baits and Chk1 and Crb3 as targets. (B) Interaction of Crb3 with the Rad4TopBP1 C terminal region containing the R3R4 BRCT domains and with Chk1 and Cds1. Two-hybrid assay with full-length Crb3 as bait and R1R2 Rad4TopBP1 and R1R2 Rad4-c11TopBP1 as targets were performed as described in Materials and Methods. R1R2 represents the construct with the deletion of R1R2 BRCT domains of rad4+TopBP1.

We further investigated whether Crb3 interacts with Chk1 and Cds1 by two-hybrid assay. Crb3 interacted with both Cds1 and Chk1 by the two-hybrid criteria (Figure 6B). Confirmation of the two-hybrid results by coprecipitation of Flag-tagged Crb3 with Cds1 or HA-tagged Chk1 from cell extracts were inconclusive due to the background affinity of Crb3 for the immuno-affinity matrix.

Crb3 Has a Role in Maintaining the Chk1 Damage Checkpoint Response
To determine whether Crb3 has a role in the Chk1 checkpoint response, we characterized crb3⁺. A strain, crb3::ura4 (crb3) in which the entire coding region of crb3⁺ gene was replaced by ura4⁺ was not viable, confirming that crb3⁺ is an essential gene (Saka et al., 1997). We then investigated the role of crb3⁺ in checkpoint response and in cell viability by generating mutants of crb3⁺ using both PCR random mutagenesis and hydroxylamine mutagenesis. Surprisingly, all of the mutants generated by either method reverted back to wild type in exceedingly high frequency.

To circumvent this difficulty, we constructed diploid strains of homozygous mating type. Diploid chk1:HA crb3/+ cells with one copy of the crb3⁺ gene deleted were tested for their sensitivity to treatment using 10 µM CPT or 5 mM HU on solid media at 30°C. Cells having a single copy of crb3⁺ exhibited higher sensitivity to CPT and HU treatment than wild type (Figure 7A), suggesting that cells with a lower crb3⁺ gene dose are more sensitive to DNA damage.

View larger version (51K): [in this window] [in a new window]   Figure 7. Effects of crb3+ gene dose on the DNA damage checkpoint response. (A) Lower crb3+ gene dose enhances cells' sensitivity to CPT and HU. Diploid cells (1 x 107) of chk1:HA +/+ (diploid with two copies of wild-type crb3+) and chk1:HA crb3/+ (diploid with one copy of crb3) were cultured to log phase, then 10-fold serial diluted, and spotted onto YES plates or plates with 10 µM CPT or 5 mM HU, followed by incubating at 30°C for 3 d. (B) Diploid wild type (+/+, with two copies of wild-type crb3+), crb3/+ (diploid cells with one copy of the crb3+ deletion), rad4-c11TopBP1/+ (diploid cells with one copy rad4-c11TopBP1 mutation), +rad4-c11TopBP1/+rad4-c11TopBP1 (diploid cells with both copies of rad4-c11TopBP1 mutation, but two copies of wild-type crb3+), and crb3 rad4-c11TopBP1/+rad4-c11TopBP1 (diploid cells with one copy of crb3+ deletion and both copies of rad4-c11TopBP1 mutation) were grown in YES media with 12 mM HU for the indicated times at 30°C, fixed, and stained with DAPI to score cells' % cut phenotype. Percentage of cut phenotype represents the average of two independent experiments of counting 300 cells per experiment. Percentage difference between the two independent points is <2.5%. (C) Fluorescence microscopy of DAPI-stained crb3 rad4-c11TopBP1/+ rad4-c11TopBP1 diploid cells after incubating in 12 mM HU for 10 h at 30°C. Arrows indicate the cells exhibiting abnormal nuclei. (D) Diploids with deletion of one copy of the crb3+ compromise the maintenance of CPT-induced Chk1 phosphorylation and Chk1 protein stability. Strains chk1:HA +/+ (diploid with HA-tagged chk1+ and both copies of wild-type crb3+) and chk1:HA crb3/+ (diploid with HA-tagged chk1+ and one copy of crb3+ deleted) were grown in YES in the presence of 30 µM CPT and incubated for 2, 6, 10, 24, and 48 h at 30°C. Chk1:HA protein was immunoprecipitated from extracts with 4 µl of rat anti-HA and protein G plus Agarose beads. Chk1 proteins were detected by immunoblotting with mouse anti-HA (12CA5). Immunoblotting of Cdc2 phosphorylation with anti-phospho-Cdc2 (Tyr15) and anti-Cdc2 (PSTAIRE) were used as an input control.

To further investigate the role of crb3⁺ in Chk1 damage checkpoint activation, we analyzed the Chk1 phosphorylation in chk1:HA crb3/+ cells containing one copy of intact crb3⁺ and compared with chk1:HA +/+ cells with both copies of crb3⁺. On CPT treatment, Chk1 protein in the chk1:HA +/+ cells was phosphorylated and remained phosphorylated up to 10 h. In the chk1:HA crb3/+ cells, the level of Chk1 protein was slightly reduced, suggesting that Chk1 also might be unstable in cells with lower crb3⁺ gene dosage. Furthermore, the CPT-induced Chk1 phosphorylation was maintained only for 6 h with no detectable Chk1 after 10 h (Figure 7D). Taken together, these results suggest that Crb3 has a role in the maintenance of Chk1 activation when DNA is damaged and may also have a role in maintaining Chk1 protein stability.

Expression of crb3⁺ Suppresses the DNA Damage Checkpoint Defect and Restores Chk1 Activation in rad4-c11^TopBP1
Finding that deletion of one copy of the crb3⁺ in rad4-c11^TopBP1/rad4-c11^TopBP1 exacerbates the damage checkpoint defect in cells prompted us to further investigate whether Crb3 influences the role of Rad4 in Chk1 damage checkpoint. We tested whether increasing the crb3⁺ gene dose by ectopic expression of crb3⁺ could suppress the CPT sensitivity of rad4-c11^TopBP1. Because activation of the Chk1 DNA damage checkpoint response depends on crb2⁺ (Saka et al., 1997; Mochida et al., 2004), crb2⁺ expression was used as a control. The cDNAs of crb3⁺ and crb2⁺ were independently constructed into the thiamine-regulated pREP41 vector (Maundrell, 1993) to express in the rad4-c11^TopBP1. Empty pREP41 vector and prad4^+TopBP1 expressed from its endogenous promoter were used as negative and positive controls, respectively, in rad4-c11^TopBP1. Cells were first grown in minimal media with thiamine and then without thiamine to induce the gene expression for 20 h. Mutant rad4-c11^TopBP1 was sensitive to 10 µM CPT. Expression of prad4^+TopBP1 from its endogenous promoter suppressed this sensitivity (Figure 8A). Significantly, moderate expression of crb3⁺ was capable in suppressing the CPT sensitivity of rad4-c11^TopBP1, indicating that an increase of crb3⁺ gene dose can somehow suppress the Chk1 damage checkpoint defect in rad4-c11^TopBP1 (Figure 8A). Interestingly, expression of crb2⁺ was also able to suppress the rad4-c11^TopBP1 CPT sensitivity, albeit to a slightly lesser extent than expression of crb3⁺. These genetic results suggest that although Crb2 and Crb3 associate with different BRCT domains of Rad4^TopBP1, the associations have a mutual influence on the function of Rad4^TopBP1 in the DNA damage checkpoint.

View larger version (46K): [in this window] [in a new window]   Figure 8. Expression of crb3+ restores DNA damage checkpoint response in rad4-c11TopBP1. (A) Expression of crb3+ suppresses the CPT sensitivity of rad4-c11TopBP1. rad4-c11TopBP1 strain was independently transformed with plasmids, pREP41 (empty vector control), prad4+TopBP1 (positive control), pREP41:crb3+, and pREP41:crb2+. Transformants (1 x 107) were serial diluted (1:10), spotted on media with or without thiamine to repress or activate the gene expression, respectively, and incubated at 30°C. The plates containing CPT did not contain thiamine. (B) Expression of crb3+ or crb2+ restores Chk1-mediated damage checkpoint in rad4-c11TopBP1. Chk1:HA was immunoprecipitated from extracts of rad4-c11TopBP1 chk1:HA cells harboring either prad4+TopBP1, pREP41:crb3+, and pREP41:crb2+ and analyzed for Chk1:HA phosphorylation by gel electrophoresis. (C) Restoration of Chk1 phosphorylation in rad4-c11TopBP1 is a specific response to DNA damage. Expression of pREP41:crb2+ or prad4+TopBP1 in undamaged rad4-c11TopBP1 did not induce Chk1 phosphorylation, whereas DNA damaged by CPT treatment-induced Chk1 phosphorylation in these cells. Cdc2 (PSTAIRE) was used as input control in all experiments.

	DISCUSSION

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

 
Studies of fission and budding yeasts have established that Rad4^TopBP1 plays an essential role in both replication and checkpoint responses (Saka and Yanagida, 1993; Araki et al., 1995; Masumoto et al., 2000). In an effort to understand how Rad4^TopBP1, a single protein, can accomplish its multiple tasks, we identified four novel mutants of rad4^+TopBP1. One mutant, rad4-c11^TopBP1, harboring an E368K substitution in the R3 domain within the conserved motif (W-X-X-X-C/S) in all BRCT domains identified, enabled us to assess the roles of Rad4^TopBP1 in checkpoint and replication, separately. A previous study of the thermosensitive mutant rad4-116^TopBP1 has shown that at its semipermissive temperature of 32°C, the mutant has viability similar to that of the wild-type cells, but loses viability after 4 h in HU, showing cut-like cells within the population after 8 h in HU. These results thus imply that the rad4-116^TopBP1 mutant lacks a DNA-replication checkpoint but not a compromised replication (McFarlane et al., 1997). This study and others of the rad4-116^TopBP1, however, have not been able to definitively and thoroughly separate the role of Rad4^TopBP1 in checkpoint responses from DNA replication by both biochemical and genetic approaches. Thus, it has been generally accepted that the roles of Rad4^TopBP1 in checkpoint and DNA replication are linked. The rad4-c11^TopBP1 mutant described in this study is defective in Chk1 activation in response to DNA damage but proficient in Cds1 activation in replication checkpoint response, DNA replication, and cell cycle progression. These results thus suggest that the acidic residue Glu³⁶⁸ plays a unique role in Chk1 activation in response to DNA damage. Thus, the role of Rad4^TopBP1 in damage checkpoint response can be separated from its roles in checkpoint response to replication perturbation and its role in DNA replication.

How Might the Mutation in rad4-c11^TopBP1 Compromise the Chk1 Damage Checkpoint Response?
Rad4^TopBP1 interacts with two proteins, Crb2 and Crb3, via the R1+R2 and R3 domains, respectively (Saka et al., 1997). Activation of Chk1 in response to damage requires Crb2 (Mochida et al., 2004). In rad4-c11^TopBP1 mutant, it is possible that the structural alteration in the R3 domain due to the E368K substitution affects the structure of R1 domain, resulting in a weakening of the interaction with Crb2 and compromising the Chk1 damage checkpoint response. Our finding that expression of crb2⁺ can restore the Chk1 activation defect in rad4-c11^TopBP1 supports this premise. A recent report has described that a crb2 mutant that is defective in Chk1 activation is able to delay the metaphase to anaphase transition via a Mad2-dependent manner when cells are exposed to CPT treatment (Collura et al., 2005). Given that deletion of chk1⁺ in rad4-c11^TopBP1 has a synergistic effect in cells sensitivity to chronic CPT treatment, it is possible that the defect of rad4-c11^TopBP1 mutation on Crb2 interaction may also compromise the Chk1-induced activation of the Mad2 -mediated spindle checkpoint to delay metaphase to anaphase transition.

Our results suggest that the mutation in rad4-c11^TopBP1 also compromises the transient interaction between Rad4^TopBP1 mutant protein with Crb3. Deletion of one copy of the crb3⁺ in diploid cells sensitizes the cells to CPT damage and compromises Chk1 maintenance. These results suggest that Crb3 has a role in Chk1 damage checkpoint. Notably, deletion of one copy of crb3⁺ gene in rad4-c11^TopBP1/+ rad4-c11^TopBP1 diploid exacerbated the cells' damage checkpoint defect after prolonged HU treatment; moderate expression of crb3⁺ restores the rad4-c11^TopBP1 mutant's Chk1 damage checkpoint response. Given the fact that Crb3 has a role in maintaining Chk1 activation and the association of Rad4^TopBP1 and Crb3 is significantly reduced in rad4-c11^TopBP1 and taking together all these findings, it is plausible that Crb3 affects the role of Rad4^TopBP1 in Chk1 damage checkpoint. However, it is not yet known how the compromised interaction between Crb3 and Rad4-c11^TopBP1 could affect the Chk1 damage defect observed in rad4-c11^TopBP1.

A recent study has shown that Chk1 activation in response to damage depends on association of Rad9 phosphorylated at T412/S423 with the R3 and R4 domains of Rad4^TopBP1 and that this association promotes coprecipitation of Rad3^ATR with Rad4^TopBP1. These findings suggest that a prerequisite for the organization of the checkpoint apparatus is phosphorylation of specific checkpoint proteins; checkpoint activation is a consequence of the interactions between the phospho-specific proteins (Furuya et al., 2004). Consistent with such a model, we show here that the level of phosphorylation of Rad9 protein at T225 is reduced in rad4-c11^TopBP1 in response to CPT-induced damage. As shown by Fuyura et al. (2004) phosphorylation at T225 of Rad9 depends on its prior phosphorylations at T412/S423. Thus, the decreased phosphorylation of Rad9-T225 reflects a reduced level of phosphorylation of Rad9 at T412/S423. Hence this could be another possible cause of the Chk1 damage response defect in rad4-c11^TopBP1.

Recent studies of TopBP1 in Xenopus egg extracts and human cells have shown that TopBP1 is a positive effector of ATR by stimulating the kinase activity of ATR for initiation of the ATR-dependent signaling processes in checkpoint responses (Kumagai et al., 2006). Rad4^TopBP1 of fission yeast associates with Rad3^ATR-Rad26^ATRIP and the rad4^TopBP1-c11 mutation does not compromise the physical association between these proteins. However, it is conceivable that although the Rad4^TopBP1-c11 mutant protein is able to associate with Rad3^ATR-Rad26^ATRIP, the rad4^TopBP1-c11 mutation somewhat compromises the ability of the mutant Rad4^TopBP1-c11 protein to stimulate the kinase activity of Rad3^ATR, thus resulting in the Chk1 damage checkpoint defect.

Rad4^TopBP1 Provides a Scaffolding Framework Linking Replication with the Checkpoint to Maintain Genomic Integrity
We show here that Rad4^TopBP1 physically associates with the checkpoint sensors and replicative polymerases, and transiently associates with Crb2 and Crb3. The three replicative polymerases, , , and , have been shown to localize and function together at replication fork throughout the cell cycle in S. cerevisiae (Hiraga et al., 2005). Findings that Rad4^TopBP1 associates with all three replicative polymerases not only during DNA replication but also during the predominately G2 phase of the fission yeast cell cycle suggests that Rad4^TopBP1 is an integral component of a protein complex at the replication fork throughout the cell cycle. The replicative polymerases are also involved in DNA repair (Holmes and Haber, 1999; Wang et al., 2004). Associations of Rad4^TopBP1 with these polymerases and checkpoint sensor proteins in G2 cells suggest that Rad4^TopBP1 may also have a role in coordinating these proteins for DNA repair.

Analyses of the four novel rad4^TopBP1 mutants show that these mutants have distinct and overlapping phenotypes. Notably, rad4-116^TopBP1 and rad4-c23^TopBP1 harboring amino acid substitutions in adjacent residues Thr⁴⁵ and His⁴⁶, respectively, have distinct phenotypes. These findings suggest that residues in each BRCT-domain of Rad4^TopBP1 could interact with different partners to enforce checkpoint response or replication. Our results in this study led us to propose that Rad4^TopBP1 serves as a scaffold to a large protein complex containing both replication proteins and checkpoint proteins. By means of its many interacting partners, Rad4^TopBP1 functions in the checkpoint machinery to monitor the genomic status and selectively activate checkpoint responses to DNA damage or replication perturbation during the cell cycle.

	ACKNOWLEDGMENTS

 
We thank A. M. Carr and K. Furuya for rad9⁺:HA strain. This work is supported by grants from the National Cancer Institute of the National Institutes of Health.

	Footnotes

 
This was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-01-0056) on May 24, 2006.

The online version of this article contains supplemental material at MBC Online (http://www.molbiolcell.org).

Address correspondence to: Teresa S.F. Wang ( tswang{at}stanford.edu)

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