BRCT Domain-containing Protein TopBP1 Functions in DNA Replication and Damage Response*

Topoisomerase II (cid:1) -binding protein (TopBP1), a human protein with eight BRCT domains, is similar to Saccharomyces cerevisiae Dpb11 and Schizosaccharo-myces pombe Cut5 checkpoint proteins and closely related to Drosophila Mus101. We show that human TopBP1 is required for DNA replication and that it interacts with DNA polymerase (cid:2) . In S phase TopBP1 colocalizes with Brca1 to foci that do not represent sites of ongoing DNA replication. Inhibition of DNA synthesis leads to relocalization of TopBP1 together with Brca1 to replication forks, suggesting a role in rescue of stalled forks. DNA damage induces formation of distinct TopBP1 foci that colocalize with Brca1 in S phase, but not in G 1 phase. We also show that TopBP1 interacts with the checkpoint protein hRad9. Thus, these results implicate TopBP1 in replication and checkpoint functions.

DNA polymerases (pol) 1 play essential roles in chromosomal DNA replication and repair. In Saccharomyces cerevisiae three essential nuclear polymerases, ␣, ␦, and ⑀ have important functions in DNA replication. S. cerevisiae pol ⑀ is isolated as a complex of a catalytic subunit and three smaller subunits, Dpb2, 3, and 4 (1). This four-subunit structure is also conserved in the human enzyme, which consists of a catalytic subunit (2), a B subunit (3,4), and two smaller subunits (5). Pol ⑀ is a proofreading DNA polymerase, which has been implicated in DNA replication, as temperature-sensitive mutants show defects in DNA replication in both S. cerevisiae and Schizosaccharomyces pombe (6 -8). Moreover, pol ⑀ is associated with origins of DNA replication and it proceeds along the replication fork (9). In human cells, pol ⑀ is associated with actively replicated cellular DNA (10) and has been shown to perform an important fraction of replicative DNA synthesis (11). Surprisingly, the catalytic domain of pol ⑀ is not essential for viability in S. cerevisiae. Instead, the C terminus, which interacts with Dpb2, exerts all of the essential functions (12).
Pol ⑀ has been proposed to function in the repair of UVdamaged DNA because it is able to catalyze UV-induced DNA synthesis in vivo (13) and performs efficient gap-filling synthesis in the reconstituted nucleotide excision repair system (14). A role in base excision repair is suggested by the fact that pol ⑀ mutants fail to support repair synthesis in vitro, and repair activity can be restored by the addition of purified pol ⑀ (15). Pol ⑀ has also been proposed to function in a specialized replication process required to repair double strand breaks (16). In addition to replicative and repair roles, it has been suggested that pol ⑀ coordinates transcriptional and cell cycle responses to DNA damage and replication blocks (17).
In S. cerevisiae, a BRCT domain-containing protein, Dpb11, interacts with the pol ⑀ complex and was originally identified as a suppressor of pol ⑀ catalytic and Dpb2 subunit mutants (18,19). DPB11 is an essential gene required for DNA replication (18). The inability of DPB11 mutants to restrain mitosis in the presence of incomplete replication suggests that Dpb11 is also needed for the replication checkpoint (18,20). Interactions of Dpb11 with Cdc45 (21) and with the checkpoint protein SLD2/ DRC1 (20,21) further link Dpb11 to the replication machinery. Apparently Dpb11 functions during early steps of DNA replication and is required for loading of DNA polymerases onto replication origins (19). Based on these observations it appears that the association of pol ⑀ with Dpb11 is important for at least part of the functions of pol ⑀.
S. pombe Cut5 also contains four BRCT domains (22,23) and is required for DNA replication as well as for both DNA damage and DNA replication checkpoints. Cut5 is required for checkpoints that prevent damaged or incompletely replicated chromosomes from being segregated (24 -27). Cut5 has been shown to associate with another BRCT domain containing protein Crb2 and with the Chk1 kinase (28).
We were interested in identifying functional homologs of budding yeast Dpb11 and fission yeast Cut5 in metazoans because of the characterized association of Dpb11 and pol ⑀. Based on homology searches we have focused our investigations on a protein with eight BRCT domains, topoisomerase II␤-binding protein (TopBP1) (29). Our results strongly suggest that the 180-kDa TopBP1 protein is required for DNA replication and strongly implicated in other functions ascribed to Dpb11 and Cut5, including checkpoint control.

EXPERIMENTAL PROCEDURES
Cloning of TopBP1 and hRad9 -A human cDNA sequence KIAA0259 (GenBank accession D87448) was used to search for human EST clones from the expressed sequence tag data base. EST clones AA013344, AA195149, and R54257 from Genome Systems, Inc. (USA) * This work was supported by grants from the Academy of Finland and from the Cancer Society of Finland. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
were used to construct full-length TopBP1 cDNA. EST clones contained deletions, and therefore nucleotides 308 -1560 and 1628 -2620 were amplified from human T-cell (HUT-78) and thymus cDNA libraries (CLONTECH). Human Rad9 cDNA was amplified from human thymus cDNA library (CLONTECH).
Generation of Antibodies-Rabbits were immunized according to standard protocols with a protein fragment representing different regions of the catalytic A subunit of human pol ⑀ or TopBP1. The antigens were prepared as described by Uitto et al. (32). The antiserum of rabbit K27 was raised against a peptide of human pol ⑀ A subunit (amino acids 1-203, GenBank accession 3192938). The rabbit antisera ␣-TopBP1.1 and ␣-TopBP1.2 were raised against amino acids 792-1003 and 1023-1167 of TopBP1, respectively. Specificity of antibodies was examined by Western analysis of human HeLa S3 cell extracts and by antigen block assay. Antibodies were purified by protein A-Sepharose CL-4B (Amersham Pharmacia Biotech).
Ribonuclease Protection Assay and Western Analysis of TopBP1-Total RNA from IMR-90 cells was isolated with TRIzol ® LS reagent (Life Technologies, Inc.). The ribonuclease protection assay was performed with an RPA III TM ribonuclease protection assay kit (Ambion) using 10 g of the total RNA. The cDNA templates for human cytoplasmic ␤-actin and histone H3 were used to prepare antisense RNA probes for ribonuclease protection assays as described (34). The template for TopBP1 was a 174-base pair fragment, representing nucleotides 2035-2209 of the cDNA. For preparation of cell extract, IMR cells were washed twice with PBS and lysed for 5 min on ice with lysis buffer (50 mM Tris-Cl, pH 7.4, 100 mM NaCl, 1% Triton X-100, 0.1% SDS, and 0.5% deoxycholic acid). Lysate was cleared by centrifugation. 20 g of protein was separated by SDS-PAGE, and ␣-TopBP1.2 antibody was used for immunoblotting.
Preparation of Permeabilized HeLa Cell Nuclei and Cytoplasmic Extracts and DNA Replication Assay in Isolated Nuclei-Preparation of HeLa S3 cell nuclei and cytoplasmic extracts and subsequent permeabilization of the nuclei with lysolecithin were performed as described by Stoeber et al. (35). Nuclei were permeabilized immediately before use, washed, and suspended by 10 strokes with a loose fitting pestle. DNA replication reactions in isolated nuclei were performed at least in triplicate per experiment as described by Pospiech et al. (11).
Cell Synchronization by Serum Starvation and the Induction of DNA Damage-IMR-90 fibroblasts were synchronized by serum starvation for 96 h as described by Tuusa et al. (34). MCF-7 cells were synchronized (36) by serum starvation for 24 h. Cells were in S phase 24 h after the readdition of serum as measured by the incorporation of [ 3 H]thymidine in parallel cell cultures (34) and punctate PCNA staining and in G 1 phase 5 h after the addition of serum. DNA was damaged (37) with 1 mM hydroxyurea (HU), 100 g/ml zeocin, 50 g/ml methyl methanesulfonate (MMS), or 10 J/m 2 UV light.
Immunostaining-Cells were grown to logarithmic phase and fixed in 3% neutral paraformaldehyde in PBS for 10 min at room temperature, and then permeabilized in 0.2% Triton X-100 in PBS for 10 min at room temperature. Alternatively, cells were fixed in 3% neutral paraformaldehyde in PBS for 10 min at room temperature and in cold methanol for either 2 or 10 min. Incubation with primary antibody was carried out for 30 min at 37°C in PBS with 0.2% gelatin (from cold water fish skin, Sigma). After washes with PBS and PBS with 0.2% gelatin, incubation with secondary antibody was carried out for 30 min at 37°C in PBS with 0.2% gelatin. DNA was stained with bisbenzimide (1 g/ml Hoechst 33258, Sigma) for 5 min at room temperature.
To label nascent DNA, MCF-7 cells were incubated in growth medium containing 0.1 mM BrdUrd (Sigma) for 3 min at 37°C. Cells were fixed with methanol-paraformaldehyde, and PCNA was immunolabeled as described above. Cells were fixed again using paraformaldehyde for 10 min and treated with 4 M HCl for 10 min to reveal incorporated BrdUrd. Immunolabeling was done as above using either fluorescein isothiocyanate-conjugated ␣-BrdUrd or ␣-BrdUrdand A-488-labeled ␣-mouse IgG. Alternatively, cells were treated with 1 mM HU for 5 h and labeled with 0.1 mM BrdUrd for 15 min right after the release.
Slides were mounted with IMMU-MOUNT (Shandon) after washes with PBS and water and examined with a fluorescence microscope (Leitz or Olympus) under the 50ϫ or 60ϫ objective. Color slides were taken using Kodak Ektachrome 400 film, or alternatively images were recorded using a CCD camera and 60ϫ or 100ϫ objective. Images were processed using Adobe Photoshop. Under the conditions used, no significant signal attributable to secondary antibody alone was detected.
Yeast Two-hybrid Assay-The BRCT domains 4 and 5 (amino acids 534 -763) of human TopBP1 were fused to LexA DNA binding domain in pLex-a vector (38). A human peripheral lymphocyte cDNA library (39) was transformed into the L40 yeast strain expressing the LexA BRCT4ϩ5 fusion. Colonies that grew on 1 mM 3-aminotriazole His Ϫ medium were assayed for ␤-galactosidase activity. Plasmids isolated from primary positives were retransformed into L40 yeast in conjunction with either pLexBRCT4ϩ5 or a number of control baits fused to LexA and tested for growth on His Ϫ medium and ␤-galactosidase activity.
Ecdysone-inducible Expression System-An Ecdysone-inducible expression system (Invitrogen) was used for overexpression of the TopBP1 and hRad9. The full-length TopBP1 cDNA was subcloned into the pIND vector and full-length hRad9 cDNA into modified pIND vector that contained FLAG epitope and SV40 T-antigen type nuclear localization signal. The linearized constructs were transfected into EcR-293 cells using FuGENE TM 6 transfection reagent (Roche Molecular Biochemicals). G418-resistant colonies were selected and cultivated further. TopBP1 expression was tested by induction with 2 M Ponasterone A for 24 h and subsequent immunoblotting. Clone TopBP1-20 showed the highest expression out of 20 G418-resistant colonies tested, with 2-3fold higher expression after induction. A similar procedure was utilized with Rad9 -5 clone. Expression levels were studied by immunoblotting with ␣-FLAG M5 antibody.
Immunoprecipitation-Subconfluent cell cultures on six-well plates were washed with PBS and suspended in 500 l of lysis buffer (100 mM Tris-Cl, pH 7.5, 100 mM NaCl, 10% glycerol, 0.1% Nonidet P-40, and protease inhibitors). Cells were disrupted by sonication for 2 ϫ 15 s (7.5-m amplitude, Soniprep 150, SANYO) on ice. After incubation on ice for 15 min, cell extract was clarified by centrifugation. Preclearing was performed with 2 l of preimmune rabbit serum. Preimmune IgG-binding proteins were collected on protein A-Sepharose. Immunoprecipitation was performed with 10 g of specific antibody. Immunocomplexes were collected on protein A-Sepharose/protein G-agarose, washed twice with lysis buffer, once with washing buffer II (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 0.5% Nonidet P-40), and once with washing buffer I (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.5% Nonidet P-40). The pellets were suspended in SDS-PAGE sample buffer for Western analysis.
Chromosomal Mapping-Chromosomal mapping of TopBP1 by fluorescence in situ hybridization (FISH) was carried out by SeeDNA Biotech Inc. (Canada) from lymphocytes isolated from human blood using a 2062-base pair cDNA probe, which was biotinylated with dATP.

RESULTS
TopBP1 Is Related to Fission Yeast Cut5, Budding Yeast Dpb11, and Drosophila Mus101-We used homology searches to identify functional human homologs of budding yeast and fission yeast Cut5 and identified a human cDNA, KIAA0259 (40), predicted to encode a protein with eight BRCT domains. This protein was identified independently in a yeast two-hybrid screen searching for DNA topoisomerase II␤-binding proteins and therefore named TopBP1 (29).
Amino acid sequence analysis revealed that the two tandem BRCT domains of Cut5 and Dpb11 are most similar to BRCT domains 1-2 and 4 -5 of TopBP1. Sequence similarities around the BRCT domains 1-2 of TopBP1 extend beyond the repeat, including amino acid residues 102-332, and this area is 35% similar (14% identical) and 44% similar (26% identical) to the N termini of Dpb11 and Cut5, respectively. In addition, the similarity around domains 4 -5 covers amino acids 547-897, where TopBP1 is 27% similar (11% identical) and 41% similar (23% identical) to the C termini of Dpb11 and Cut5, respectively. However, TopBP1 contains additional BRCT domains, which are not present in either Dpb11 or Cut5 as shown schematically in Fig. 1. TopBP1 is not able to suppress temperature-sensitive mutants of either budding yeast DPB11 (dpb11-1) or fission yeast Cut5 (cut5-T401 or cut5-580) (data not shown; see "Experimental Procedures").
Nevertheless, the observed similarities imply that TopBP1 may be executing some tasks similar to those performed by the yeast proteins. This notion is supported by the recent identification of the apparent Drosophila TopBP1 homolog, which is encoded by the mus101 locus (41). mus101 has been genetically linked to DNA synthesis (42), DNA repair, and chromosome condensation at mitosis (43,44). The predicted Mus101 protein contains seven BRCT domains, and the overall similarity with human TopBP1 is 38% (28% identity; see Fig. 1). Another apparent homolog is present in the nematode Caenorhabditis elegans, where the predicted F37D6.1 protein contains six BRCT domains as three tandem repeats (Fig. 1) and shares 38% similarity (28% identity) with the N terminus of TopBP1 (amino acids 15-470) and 47% similarity (33% identity) with the C-terminal amino acids 1287-1465 of TopBP1, respectively. This homolog is not well characterized, however.
The C-terminal Region of TopBP1 Is Similar to Brca1, and TOPBP1 Localizes to 3q21-q23-In addition to the homologies described above, the C-terminal region of TopBP1 containing two BRCT domains shows similarity to the C-terminal region of the breast cancer susceptibility gene product Brca1. Amino acids 1014 -1522 of TopBP1 share 35% similarity and 25% identity to amino acids 1335-1863 of Brca1, corresponding to the two C-terminal BRCT domains as well as the region upstream of the domains in Brca1. This region of Brca1 is important for many of the functions of Brca1, including tumor suppressor activity (45). Therefore we investigated whether the genomic locus of the TOPBP1 gene was a target for chromosomal abnormalities in human cancers. To this end we assigned the human TOPBP1 gene to chromosome 3, region q21-q23 by FISH (Fig. 2) using a 2 kilobase cDNA probe obtained from EST clone AA013344. This chromosomal region, however, has not been associated with disease genes of frequent abnormalities in human cancers according to the OMIM data base.
TopBP1 Is a 180-kDa Phosphoprotein with Highest Expression in the S Phase-To characterize the TopBP1 protein, we raised polyclonal rabbit antibodies against GST-TopBP1 fusion proteins. Two independent rabbit antisera, ␣-TopBP1.1 and ␣-TopBP1.2, specifically recognized a polypeptide from HeLa cell extracts with an apparent molecular mass of 180 kDa (Fig.  3A), which is slightly larger than the calculated molecular mass of 171 kDa. This difference may be caused by phosphorylation, as in vivo phosphate labeling indicated that TopBP1 is a phosphoprotein (Fig. 3B). The phosphorylation of TopBP1 occurred mostly on serine and to a lesser extent on threonine

TopBP1 in DNA Replication and Damage Response
residues according to phosphoamino acid analysis (data not shown). No labeling was detected on tyrosine residues.
We also studied TopBP1 expression levels in IMR-90 fibroblasts re-entering the cell cycle after serum starvation. Western blotting analysis indicated that the steady-state levels of TopBP1 started to increase 14 h after restimulation with serum (Fig. 3C). The increase in TopBP1 protein levels is likely to be a result of increased transcription because RNase protection assays indicated a corresponding increase in TOPBP1 mRNA levels in a parallel experiment (Fig. 3D).
After a peak at 22 h, the mRNA levels decreased again as detected in the 26-and 30-h samples. This indicates that TopBP1 expression is induced concomitantly with S phase entry as demonstrated by thymidine incorporation analysis (Fig. 3E). The induction is not specific to the first cycle after a cell cycle arrest because a similar S phase peak was observed in HeLa S3 cells, which were first synchronized at M phase with nocodazole and then released from the block (data not shown). These results indicate that TopBP1 expression is highest in S phase cells, suggesting that TopBP1 may be involved in DNA replication.
TopBP1 Is Required for DNA Replication-The results presented above together with the requirement of both Cut5 and Dpb11 for DNA replication prompted us to investigate whether TopBP1 is required for chromosomal DNA replication in human cells. Replicative DNA synthesis in isolated HeLa cell nuclei was measured by incorporation of radioactive dCMP into newly synthesized DNA (46). One of our polyclonal anti-TopBP1 antisera, ␣-TopBP1.1, efficiently inhibited replicative DNA synthesis by 44% (Fig. 4), whereas the preimmune serum or another TopBP1 antiserum, ␣-TopBP1.2, did not inhibit DNA synthesis. The inhibition by ␣-TopBP1.1 was almost as efficient as inhibition by neutralizing antibodies against human DNA polymerase ␣ and ⑀ employed previously in this assay (11).
Interestingly, DNA replication could be inhibited even more dramatically (83%) by adding recombinant TopBP1-792-1003 protein to the reaction. This fragment contains the sixth BRCT domain and was also the antigen used to generate ␣-TopBP1.1. No inhibitory effect was detected when using an adjacent fragment TopBP1-1023-1167, which was used as the antigen for ␣-TopBP1.2. From these results we conclude that TopBP1 is required for replication of chromosomal DNA.
TopBP1 and Brca1 Colocalize in S Phase-To investigate further the role of TopBP1 in DNA replication we tested whether TopBP1 is localized to sites of DNA synthesis. For this purpose we first confirmed that the BrdUrd incorporation pattern overlies the immunostaining pattern of PCNA (Fig. 5A). Inhibition of replicative DNA synthesis by ␣-TopBP1.1 and TopBP1-792-1003 is shown. Replicative DNA synthesis using isolated nuclei and cytoplasmic extract was measured by incorporation of radioactive dCMP into newly synthesized DNA (11). Efficient incorporation of nucleotides only takes place in the presence of nuclei and cytoplasmic extract (CyE). The indicated antibodies or purified fragments of TopBP1 (100 g/ml, except with ␣-TopBP1.1, where 20, 50, or 100 g/ml was used as indicated) were incubated for 90 min on ice with reaction mixture prior to replication reaction. Bars represent the incorporation of dCMP as a percentage of dCMP incorporation in complete reaction without additions, and error bars show standard deviation of triplicate samples.

TopBP1 in DNA Replication and Damage Response
We then subjected S phase MCF-7 cells (see "Experimental Procedures") to double labeled immunofluorescence analysis with ␣-TopBP1.2 and ␣-PCNA as a marker for sites of DNA synthesis. The TopBP1 signal was detected primarily in the nucleus of MCF-7 cells where it localized to several distinct nuclear foci. PCNA was also mostly detected as nuclear foci as expected, but interestingly, the TopBP1 foci were distinct from these as demonstrated by an overlay of the two (Fig. 5B). This indicates that most of TopBP1 does not colocalize with sites of ongoing DNA replication. This pattern is similar to that of Brca1 (47), and therefore we analyzed whether TopBP1 and Brca1 foci overlap. Concomitant staining with polyclonal TopBP1 and monoclonal Brca1 antibodies demonstrated colocalization of these two proteins (Fig. 5C). All foci detected with anti-Brca1 were also positive for TopBP1, but a number of additional TopBP1 foci were detected. A similar pattern of TopBP1 staining was also noted in IMR-90 fibroblasts (data not shown).
Replication Blocks Target TopBP1 and Brca1 to Stalled Replication Forks-We also analyzed the localization of TopBP1 in response to replication blocks, which were induced by treating MCF-7 cells with HU for 5 h. Examination of cells that were released from the HU block revealed that TopBP1 now colocalized with sites of BrdUrd incorporation (Fig. 6A). Similarly, in HU-treated synchronized S phase cells TopBP1 also colocalized with PCNA (Fig. 6B). These results suggest that in response to replication blocks TopBP1 is rapidly relocalized to foci representing stalled replication forks. A similar behavior has also been reported for Brca1 (47), and accordingly, the colocalization of TopBP1 and Brca1 was maintained after the HU block (Fig. 6B).
Localization of TopBP1 after DNA Damage in S Phase-We also tested the effect of several DNA damaging agents on the localization pattern of TopBP1 during S phase. S phase MCF-7 cells were treated with a UV pulse of 10 J/m 2 and analyzed 5 h later. UV irradiation induced formation of numerous bright TopBP1 foci, which were shown by double staining to colocalize uniformly with Brca1 (Fig. 6C). Colocalization with PCNA was significantly lower than after HU treatment and represented 19% of TopBP1 foci (Fig. 6, C and E). Therefore the translocation of TopBP1 is not solely a consequence of arrested replica-tion forks. Double strand breaks, created by addition of the ␥-radiation-mimicking reagent zeocin, also induced formation of bright TopBP1 foci that colocalized with Brca1 ( Fig. 6D) but not with PCNA (Fig. 6, D and E). A less pronounced focus formation was observed when we treated cells with an alkylating agent, MMS, which induced an increased number of TopBP1 foci that colocalized with Brca1 (data not shown) and to a small extent with PCNA (Fig. 6E). Taken together, we show that various types of DNA-damaging agents induce formation of bright TopBP1 foci, which are more abundant than in undamaged cells. These foci coincide with Brca1 foci, suggesting that during S phase TopBP1 and Brca1 function in related DNA damage-responsive pathways.
Double Strand Breaks Recruit TopBP1 to G 1 Foci-We also addressed the question of whether the focal accumulation of TopBP1 to new foci in response to DNA damage and replication blocks is a cell cycle-specific phenomenon using synchronized MCF-7 cells. In untreated G 1 phase cells, TopBP1 exhibits rather uniform nuclear and to a lesser extent cytoplasmic staining (Fig. 6, F and H). After zeocin treatment to induce double strand breaks, TopBP1 was relocalized to a large extent into foci (Fig. 6, G and H). The G 1 focus formation was not as clearly detected with HU, UV, or MMS (Fig. 6H). The results indicate that relocalization of TopBP1 after DNA damage is not specific for S phase and can be detected independently of Brca1, as the Brca1 signal was weak in the G 1 cells as reported earlier (36).
TopBP1 Interacts with Human Checkpoint Protein hRad9 -The presence of multiple BRCT domains in TopBP1 suggested the possibility of protein-protein interactions. To investigate this we initially undertook two-hybrid interaction screening. Full-length TopBP1 as well as the tandem BRCT domain repeats 1-2 fused to the DNA binding domain LexA activated the lacZ reporter gene, and therefore we used BRCT domains 4 -5 to screen a human peripheral lymphocyte cDNA library for interacting clones. One of the interacting clones contained the C-terminal 148 amino acids of human Rad9 fused to GAL4. The specificity of the interaction was shown further by the failure of hRad9 to form two-hybrid interactions with the BRCT domains 3 and 6 of TopBP1 (Fig. 7A) or several other partners, such as lamin, pol ⑀, Cdc45, and Chk1 (data not shown). The hRad9-TopBP1 interaction was also detected using a full-length hRad9 LexA fusion and full-length TopBP1 expressed as a VP16 activation domain fusion (Fig. 7A). To verify the interaction between cellular TopBP1 and hRad9, we used EcR-293 cells stably transfected with a Ponasterone A-inducible FLAGtagged hRad9 (Fig. 7B). When anti-FLAG immunoprecipitates from these cells were probed for the presence of endogenous TopBP1, a 180-kDa band was detected specifically in cells induced to overexpress hRad9 (Fig. 7C). These results indicate that TopBP1 and hRad9 interact in vivo via the BRCT domains 4 -5 of TopBP1.
TopBP1 Interacts with Human DNA Polymerase ⑀-Because DNA pol ⑀ interacts with Dpb11 both genetically and physically (18,19) in the budding yeast, we investigated the possible association of TopBP1 with human pol ⑀. TopBP1.2 and control immunoprecipitates from EcR-293 cell lysates overexpressing TopBP1 were probed with a pol ⑀ antibody. A Ͼ 220 kDa band was specifically detected only in the TopBP1.2 immunoprecipitates (Fig. 7D), indicating the association of endogenous pol ⑀ with TopBP1. This interaction could also be detected in HeLa S3 cell lysates (data not shown). We did not detect human pol ␣ or pol ␦ in TopBP1 immunoprecipitates, nor could we immunoprecipitate pol ⑀ together with TopBP1 under denaturing conditions (data not shown).
The interaction of TopBP1 with pol ⑀ is not cell cycle-dependent because TopBP1 and pol ⑀ coimmunoprecipitated in all

TopBP1 in DNA Replication and Damage Response
phases of the cell cycle from HeLa cells fractionated by centrifugal elutriation (data not shown). DNA damage or replication blocks did not affect the association because no effect in coimmunoprecipitation was observed after UV damage, MMS, or HU treatment (Fig. 7E). DISCUSSION The results presented here implicate the BRCT domaincontaining protein TopBP1 in DNA replication and damage responses, functions ascribed to both budding yeast Dpb11 and fission yeast Cut5. Our initial observations of the similarity of

TopBP1 in DNA Replication and Damage Response
the four BRCT domains of TopBP1 (KIAA0259) with Dpb11 and Cut5, together with the requirement for DNA replication and relocalization in response to DNA damage, support the idea that TopBP1 shares functional characteristics with Dpb11 and Cut5. The presence of six BRCT domains in the apparent nematode C. elegans homolog implies that the metazoan homologs of the yeast proteins have acquired additional domains and functions. The recent identification of the Drosophila Mus101 locus encoding an apparent Drosophila homolog of TopBP1 (41) strongly corroborates the notion of functional similarities between TopBP1/Mus101 and Dpb11 and Cut5. Mus101 is essential for viability, and different alleles of mus101 display phenotypes caused by defects in normal DNA synthesis, mutagen sensitivity, and defects in postreplication repair. Some mus101 mutants also exhibit chromosomal instability and have undercondensed or broken chromosomes (41,48).
The recruitment of TopBP1 to stalled replication forks after inhibition of replication suggests that TopBP1 may be important in the rescue of stalled forks. In S. cerevisiae, the Dpb11⅐pol ⑀ complex associates with sites of initiation of chromosomal replication, and this association seems to be required for loading of pol ␣-primase (19). Like Dpb11, TopBP1 also interacts physically with pol ⑀, and possibly TopBP1 also has a similar role in loading of essential replication factors in initiation or early steps of DNA replication. Our results suggest that the BRCT domain 6 of TopBP1 interacts with a critical replication factor, given the strong inhibition of DNA replication we observed with both ␣-TopBP1 antibody and the antigen. It will be of interest to identify the protein(s) that interact with this domain. Although our results do not address the function of TopBP1 after initiation of replication, it has been proposed earlier that Brca1 is localized to sites specialized for the processing of replicating or replicated DNA (47). The observed colocalization of TopBP1 and Brca1 together with the requirement of TopBP1 for replication reinforced this hypothesis and suggests that also TopBP1 may be involved in these functions.
In addition to the replicative role, TopBP1 is also implicated in DNA damage response as it relocates together with Brca1 to distinct foci in S phase. Based on the interaction of TopBP1 with hRad9, these foci could harbor, in addition to TopBP1 and Brca1, hRad9 and its associated proteins Hus1 and Rad1 (49 -51). Interestingly, Rad9, Rad1, and Hus1 seem to be structurally related to PCNA (52)(53)(54), and these proteins have been shown to interact with the human checkpoint protein hRad17, which is predicted to resemble replication factor C, RFC, structurally (55). During the DNA damage response, the PCNArelated checkpoint proteins might form clamp-like structures that participate in the recognition or processing of damaged DNA as suggested earlier by Volkmer and Karnitz (50). The BRCT domains of TopBP1 have been shown to bind DNA breaks (56), and this together with our observations raises the possibility that TopBP1 might be involved in damage recognition.
The colocalization of Brca1 and TopBP1 before and after DNA damage and replication blocks suggests that in S phase, TopBP1 and Brca1 function in a related pathway. However, we have been unable to coimmunoprecipitate these proteins, indicating that although TopBP1 and Brca1 are recruited to the same subcellular sites, they do not physically interact. The fact that TopBP1 interacts with both hRad9 and pol ⑀ and is also able to bind DNA breaks (56) is suggestive of a role as a damage sensor and would be consistent with TopBP1 being upstream of the damage kinases and BRCA1 in the DNA damage pathway. It was shown recently that in human cells, hCds1 phosphorylates, interacts, and colocalizes with Brca1 (57). Brca1 has also been linked to the protein kinase ataxia telangiectasia mutated (ATM), which phosphorylates Brca1 after double strand breaks in chromosomal DNA (58) and more recently to ATM and Rad3-related (ATR), which phosphorylates Brca1 in response to UV irradiation and DNA replication inhibitors (59). Our whole cell extracts of stably transfected EcR293 cells induced to overexpress TopBP1 with 2 M Ponasterone A were subjected to immunoprecipitation with ␣-pol ⑀ (K-27), ␣-TopBP1.2, and ␣-mouse IgG rabbit antibodies, separated by SDS-PAGE, and immunoblotted by using a mixture of ␣-pol ⑀ antibodies (H3B, G1A, and E24A, 2 g/ml each). The asterisk denotes an approximately 180-kDa pol ⑀ that is consistently recognized in ␣-TopBP1.2 immunoprecipitates by N-terminal ␣-pol ⑀ antibodies H3B and G1A, but not by C-terminal E24A (data not shown). The far right lane is a whole cell extract. Panel E, association of TopBP1 with pol ⑀ after DNA damage by 15 J/m Ϫ2 UV, 2 mM HU, or 50 g/ml MMS was monitored as in panel D. C denotes undamaged control sample. attempts to establish links between TopBP1 and the checkpoint kinases hChk1 or hCds1 by the two-hybrid system or immunoprecipitation have failed, and we observed no difference in de novo phosphorylation of TopBP1 or in tryptic phosphopeptide maps after UV irradiation, zeocin, MMS, or HU (data not shown). Therefore, it seems likely that although TopBP1 is a phosphoprotein in vivo, it is not a substrate for DNA damage-responsive kinases. This is coherent with the assumption that TopBP1 acts early in the DNA damage-responsive pathway. Interestingly, although TopBP1 and Brca1 have similar responses to genotoxic insult during S phase, TopBP1 also responds to DNA damage independent of Brca1 in G 1 phase, indicating that TopBP1 plays an as yet unidentified role in the cellular response to double strand breaks in G 1 phase.
Taken together, TopBP1 as a protein that physically interacts with the components of DNA replication and DNA damage checkpoint machinery and with functions in DNA replication and DNA damage response, might play crucial roles in the integration of DNA replication and DNA damage signal transduction pathways.