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• W2046003114 abstract "A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment. A growing body of evidence suggests that establishment of sister chromatid cohesion is dependent on replication fork passage over a precohesion area. In Saccharomyces cerevisiae, this process involves an alternative replication factor C (RFC) complex that contains the four small RFC subunits as well as CTF18, CTF8, and DCC1. Here, we show that an evolutionarily conserved homologous complex exists in the nucleus of human cells. We demonstrate that hCTF18, hCTF8, and hDCC1 interact with each other as well as with the p38 subunit of RFC. This alternative RFC-containing complex interacts with proliferating cell nuclear antigen but not with the Rad9/Rad1/Hus1 complex, a proliferating cell nuclear antigen-like clamp involved in the DNA damage response. hCTF18 preferentially binds chromatin during S phase, suggesting a role during replication. Our data provide evidence for the existence of an alternative RFC complex with a probable role in mammalian sister chromatid cohesion establishment. Sister chromatid cohesion plays an integral role in the maintenance of genomic stability. Cohesion proteins help prevent premature sister chromatid separation and facilitate the correct orientation of chromatids on the metaphase plate. This allows the kinetochore of each chromatid to capture microtubules emanating from opposite poles of the mitotic spindle. During a normal cell division, the cohesive force prevents premature separation of sisters by opposing the splitting force of the spindle. The correct chromosomal alignment and balance of tensions send a signal to the mitotic spindle checkpoint. This permits activation of the metaphase-anaphase transition and results in segregation of each sister chromatid to the appropriate daughter cell (1Cohen-Fix O. Cell. 2001; 106: 137-140Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 2Lee J.Y. Orr-Weaver T.L. Annu. Rev. Dev. Biol. 2001; 17: 753-757Crossref PubMed Scopus (139) Google Scholar, 3Nasmyth K. Annu. Rev. Genet. 2001; 35: 673-745Crossref PubMed Scopus (592) Google Scholar, 4Jallepalli P.V. Lengauer C. Nat. Rev. Cancer. 2001; 1: 109-117Crossref PubMed Scopus (339) Google Scholar). As expected, abnormalities in sister chromatid cohesion interfere with this process, resulting in an increased rate of aneuploidy in daughter cells (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 6Hanna J.S. Kroll E.S. Lundblad V. Spencer F.A. Mol. Cell. Biol. 2001; 21: 3144-3158Crossref PubMed Scopus (263) Google Scholar, 7Naiki T. Kondo T. Nakada D. Matsumoto K. Sugimoto K. Mol. Cell. Biol. 2001; 21: 5838-5845Crossref PubMed Scopus (99) Google Scholar). Although the role of cohesion in the metaphase-anaphase transition is relatively well understood, there is comparatively little information about the establishment of cohesion. It is known that this process occurs during S phase and presumably is linked to the replication fork (1Cohen-Fix O. Cell. 2001; 106: 137-140Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 2Lee J.Y. Orr-Weaver T.L. Annu. Rev. Dev. Biol. 2001; 17: 753-757Crossref PubMed Scopus (139) Google Scholar, 3Nasmyth K. Annu. Rev. Genet. 2001; 35: 673-745Crossref PubMed Scopus (592) Google Scholar, 4Jallepalli P.V. Lengauer C. Nat. Rev. Cancer. 2001; 1: 109-117Crossref PubMed Scopus (339) Google Scholar). In addition, it is thought to involve a polymerase switch analogous to the one mediated by the replication factor C (RFC) 1The abbreviations used are: RFC, replication factor C; CTF, chromosomal transmission fidelity; DCC, defective in sister chromatid cohesion; PCNA, proliferating cell nuclear antigen, SMC, structural maintenance of chromosomes; PBS, phosphate-buffered saline. complex during lagging strand DNA synthesis (10Carson D.R. Christman M.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8270-8275Crossref PubMed Scopus (65) Google Scholar). RFC functions by loading the processivity factor PCNA onto DNA and mediating the switch from DNA polymerase α to DNA polymerase δ. A novel DNA polymerase, DNA polymerase σ (8Wang Z. Castano I.B. De Las Penas A. Adams C. Christman M.F. Science. 2000; 289: 774-779Crossref PubMed Scopus (163) Google Scholar), initially named DNA polymerase κ (9Burgers P.M.J. Koonin E.V. Bruford E. Blanco L. Burtis K.C. Christman M.F. Copeland W.C. Friedberg E.C. Hanaoka F. Hinkle D.C. Lawrence C.W. Nakanishi M. Ohmori H. Prakash L. Prakash S. Reynaud C.-A. Sugino A. Todo T. Wang Z. Weil J.-C. Woodgate R. J. Biol. Chem. 2001; 276: 43487-43490Abstract Full Text Full Text PDF PubMed Scopus (284) Google Scholar), has recently been described. This novel polymerase is thought to mediate DNA synthesis over areas bound to cohesion proteins, but the mechanism of recruitment of this polymerase is unknown. Presumably, DNA polymerase σ relocates to replication forks with the aid of RFC or a related clamp loader complex. This alternative clamp loader may be a recently described complex consisting of RFC2–5 and CTF18/Chl12, identified in Saccharomyces cerevisiae (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 6Hanna J.S. Kroll E.S. Lundblad V. Spencer F.A. Mol. Cell. Biol. 2001; 21: 3144-3158Crossref PubMed Scopus (263) Google Scholar, 7Naiki T. Kondo T. Nakada D. Matsumoto K. Sugimoto K. Mol. Cell. Biol. 2001; 21: 5838-5845Crossref PubMed Scopus (99) Google Scholar). Two additional proteins, CTF8 and DCC1, were also shown to interact with CTF18 in this complex (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). It has been hypothesized that CTF18, CTF8, and DCC1 form part of a clamp loader complex that tethers an unidentified clamp to precohesion areas of DNA, resulting in the recruitment of DNA polymerase σ. The passage of polymerase σ over the precohesion area coincides with cohesion establishment, suggesting that the replication fork plays an active role in this process (10Carson D.R. Christman M.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8270-8275Crossref PubMed Scopus (65) Google Scholar). There is evidence that polymerase σ recruitment depends on the CTF18/CTF8/DCC1-containing complex. In yeast, mutation of any of these three proteins resulted in precocious sister chromatid separation, increased rates of chromosome loss, and synthetic lethality with mutations in cohesion proteins such as SCC1 and SMC3 (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar, 6Hanna J.S. Kroll E.S. Lundblad V. Spencer F.A. Mol. Cell. Biol. 2001; 21: 3144-3158Crossref PubMed Scopus (263) Google Scholar, 7Naiki T. Kondo T. Nakada D. Matsumoto K. Sugimoto K. Mol. Cell. Biol. 2001; 21: 5838-5845Crossref PubMed Scopus (99) Google Scholar). Here, we describe the cloning and the initial characterization of this evolutionary conserved alternative RFC complex in Homo sapiens. The three proteins, which we designate hCTF18, hCTF8, and hDCC1, show the same pattern of association in humans as in S. cerevisiae. This suggests a high degree of conservation between yeast and humans in the machinery of cohesion establishment. Predicted Protein Features and Homology—scCTF18 (NP_013795.1), scCTF8 (NP_012061), and scDCC1 (NP_009913) sequences were used to identify human homologs by BLAST search (NCBI). The DNA sequences were translated into hypothetical proteins, whose theoretical characteristics were obtained using several programs in the ExPASy (Expert Protein Analysis System) server of the Swiss Institute of Bioinformatics (www.expasy.ch/tools/). The protein sequences were entered into MotifScan (pattern searches), PSORT (sorting and localization signal identification), and ProtParam (chemical features). Yeast and human CTF18 amino acid sequences were aligned using the PileUp feature of WebSeq, available at mutant.mayo.edu. Antibodies—We used two complementary methods for antibody production. Most of our antibodies were obtained from synthetic peptides selected for maximum antigenicity. These peptide sequences were CKRTRDEVDATLQIAKLNAAE (hDCC1-N), CQNGVKVYNSRRPIS (hDCC1-C), CKDKILFKTRPKPIITSVPKKV (hCTF8-C), EEMEEPPPPDSSPTDITPPPSPEDC (hCTF18-A), HERPSRKDRPSVEPARVSKEC (hCTF18-C), CAPRNHEQRLEHIMRRAAREEQPEK (hCTF18-D), CNIQQKTDEKVDESGPPAPSK and CPRRGRRPKSESQGNATKND (hPds5); CKRKRGRPGRPPSTNKKPRKS and CSSSSKTSSVRNKKGRPPLHKKR (SA1); and CSSRGSTVRSKKSKPSTGKRKVV and CDLPPSKNRRERTELKPDFFD (SA2). Solutions of these peptides coupled to mariculture keyhole limpet hemocyanin (Pierce) were used for rabbit polyclonal antibody production. Antibodies recognizing hDCC1 (αhDCC1–4, αhDCC1–5, and αhDCC1–6) were produced from different glutathione S-transferase-fused DCC1 protein fragments. hDCC1 cDNA was amplified via reverse transcription-PCR (Titan System; Roche Applied Science) from RNA isolated from HBL-100 cells. The sequences were subcloned into a pGEX-4T-1 vector, which was then transformed into competent BL21 codon + cells. Following induction of BL21 codon + cells with 0.4 mm isopropyl-β-d-thiogalactopyranoside, glutathione S-transferase fusion proteins were bound to glutathione-conjugated Sepharose beads and eluted with 5 mm glutathione. Eluates were used for rabbit polyclonal antibody production following standard procedure. All of the antibodies were affinity-purified with protein/peptide-conjugated Sepharose bead columns. Immunoprecipitation—The cells were harvested, washed with PBS, and lysed with 1× NETN buffer (100 mm NaCl, 1 mm EDTA, 20 mm Tris-HCl, pH 8.0, 0.5% Nonidet P-40) at 4 °C for 10 min. Some experiments were repeated using a gentler lysis buffer (11Rauen M. Burtelow M.A. Dufault V.A. Karnitz L.M. J. Biol. Chem. 2000; 275: 29767-29771Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar) containing 10 mm HEPES, pH 7.4, 150 mm KCl, 10 mm MgCl2, and 0.1% Triton X-100. For immunoprecipitation, 200 μl of whole cell lysate was incubated with 2 μg of antibody and 20 μl of protein A-Sepharose beads (Sigma) on a shaker at 4 °C for 90 min. Following three washes with lysis buffer, the immunoprecipitates were fractionated via SDS-PAGE (10% gel). The proteins were then transferred to an Immobilon-P™ polyvinylidene difluoride membrane (Millipore, Bedford, MA). The membranes were incubated with 1:1000 dilutions of primary antibody and 1:1000 dilutions of protein A-horseradish peroxidase and were developed using SuperSignal chemoluminescent solution (Pierce). Selected membranes were stripped with 7 m guanidine hydrochloride for 30 min and reblotted. Cell Culture and Synchronization—MCF-7, T24, and HeLa cells were cultured in 10-cm plates with either Dulbecco's modified Eagle's medium or RPMI 1640 medium containing 10% fetal calf serum and 1% 10,000 units/ml of penicillin/streptomycin. T24 cells were synchronized by contact inhibition. They were released into the cell cycle after 4 days by subculturing in a 1:4 ratio and harvested at selected time points. HeLa cells were synchronized by drug treatment. S phase fractions were obtained either by a 24-h treatment with 1 mm hydroxyurea (Sigma) or a double block with 2 mm thymidine (Sigma). The cells were treated with thymidine for 17 h, given fresh medium for 10 h, retreated with thymidine for 19 h, and then harvested. M phase fractions were obtained either by treatment with 10 ng/ml nocodazole (Sigma) or by agitating plates of normally cycling cells and harvesting the nonadherent mitotic fraction. In all cases, cell cycle distribution was verified by fluorescence automated cell sorting. Immunofluorescence—HeLa cells were grown to 20% confluence on glass slides affixed to the bottom of 6-well plates. They were fixed with 3% paraformaldehyde for 20 min at 4 °C, permeabilized with 1% Triton X-100 with 0.5% Nonidet P-40 for 30 min, and blocked with 5% milk in PBS for a minimum of 3 h at 4 °C. The cells were then incubated in affinity-purified primary antibody (αPC-10 PCNA and either αhCTF18-D or αhDCC1-N) and 5% goat serum with PBS for4hat4 °C. Immune complexes were detected with rhodamine-conjugated goat anti-rabbit (Jackson) and fluorescein isothiocyanate-conjugated goat anti-mouse IgG (Jackson) in 5% goat serum with PBS. Chromatin was counterstained with 2 μg/ml Hoechst 33342 in PBS for 30 s. Stained cells were analyzed with a Nikon ECLIPSE E800 fluorescence microscope. Cell Fractionation—To verify subcellular CTF18 localization and to determine the affinity of CTF18 for chromatin at different stages of the cell cycle, HeLa cells were fractionated using a protocol modified slightly from Méndez and Stillman (13Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (755) Google Scholar). HeLa cells were synchronized as described above, harvested, and resuspended in PBS to equalize cell concentration. The cells were first treated with a nonionic detergent (10 mm HEPES, pH 7.9, 10 mm KCl, 1.5 mm MgCl2, 0.34 m sucrose, 1 mm dithiothreitol, 10% glycerol, and 0.1% Triton X-100 supplemented with a protease inhibitor mixture (Roche Applied Science)) for 5 min on ice to isolate nuclei. Nuclei were spun down at 1300 × g for 4 min at 4 °C, and the supernatant (cytoplasmic fraction) was clarified at 20,000 × g for 20 min at 4 °C. Isolated nuclei were either directly lysed with nuclear lysis buffer (3 mm EDTA, 0.2 mm EGTA, 1 mm dithiothreitol, and protease inhibitors), or pretreated with 1 unit of micrococcal nuclease to release chromatin-bound proteins. Nuclease treatment was performed in nuclear isolation buffer supplemented with 1 mm CaCl2 and micrococcal nuclease (Sigma) for 1 min at 37 °C. The reaction was quenched with 1 mm EGTA. The supernatant from this reaction was then clarified by high speed centrifugation. Untreated nuclei were lysed with nuclear lysis buffer for 30 min on ice and centrifuged for 5 min at 3000 × g. The nuclear lysate was clarified with high speed centrifugation. The Alternative RFC Complex Containing CTF8, CTF18, and DCC1 Is Evolutionarily Conserved—A BLAST search of the NCBI data base revealed candidate human homologs of S. cerevisiae CTF8, CTF18, and DCC1. Relevant GenBank™ accession numbers are BC018700 (hCTF8), BC006437 (hCTF18), and BC001316 (hDCC1). We translated these sequences into hypothetical proteins and compared them with the known sequences of S. cerevisiae CTF8, CTF18, and DCC1. Overall amino acid homologies between S. cerevisiae and humans are 27.7% (CTF18), 29.7% (CTF8), and 30.0% (DCC1), with areas of much greater homology for certain conserved domains. We analyzed the amino acid sequences of hCTF18, hCTF8, and hDCC1 with various pattern search programs to determine whether their structural motifs might provide insight into protein function. hDCC1 does not contain any known functional domains. However, the alignment of human and S. cerevisiae DCC1 reveals two areas with over 50% conservation, which may correspond to novel domains. One is N-terminal (consensus sequence DKDXXXXVLCSXDKTXXLK), and the other is C-terminal (consensus sequence LPXDXXERFXXLFX- LXXXWXXEDIXPXI). The motifs present in hCTF18 indicate a structural similarity to clamp loader proteins such as RFC1 and Rad17. These include an RFC conserved domain and an NTP-binding motif/NTPase domain (Fig. 1). The RFC conserved domain is also present in yeast CTF18/Chl12, all five subunits of replication factor C and its homolog AC1 (eukaryotic activator 1), Schizosaccharomyces pombe Rad17/S. cerevisiae Rad24, Drosophila germline transcription factor 1 (GFN1), and two subunits of bacterial DNA polymerase III. It is thought that this domain mediates DNA binding. In addition, two leucine zipper motifs of unknown significance are present in the C-terminal region of hCTF18 but not in its S. cerevisiae homolog. Structural motif searches of hCTF8 reveal another leucine zipper, which has been noted previously by Mayer et al. (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). hCTF18, hCTF8, and hDCC1 Form a Complex in the Cell—To confirm that these putative human homologs of yeast CTF8/CTF18/DCC1 do form a complex in mammalian cells, we generated multiple rabbit polyclonal antibodies against hCTF18, hCTF8, and hDCC1 (Fig. 2A). hDCC1 immunoprecipitates contained a band migrating at ∼45 kDa, which is consistent with the predicted molecular mass for hDCC1 of 44.8 kDa. hCTF18 immunoprecipitates contained a band near 110 kDa, consistent with the theoretical molecular mass for hCTF18 of 110.2 kDa. To determine whether these three proteins interact in humans, we immunoprecipitated MCF-7 whole cell lysates with αhDCC1-C, αhCTF18-C, and αhCTF8-C and immunoblotted with αhCTF18-D or αhDCC1-N antibodies. As shown in Fig. 2B, all three proteins associated with each other, suggesting that a complex containing hCTF18, hCTF8, and hDCC1 does exist in human cells. The Expression of hCTF18 and hDCC1 Is Cell Cycle-regulated—Because hCTF18, hCTF8, and hDCC1 may be involved in cohesion establishment during S phase in humans, we examined whether the expression of these proteins might be regulated in a cell cycle-dependent manner. We analyzed hDCC1 and hCTF18 protein levels in T24 cells that were grown to confluence (arrested by contact inhibition at G0 phase). After 4 days, the cells were trypsinized, replated in fresh medium, and harvested at various time points. As shown in Fig. 3, hDCC1 and hCTF18 protein levels are undetectable at 0 and 8 h after replating. In contrast, the proteins are expressed at low levels as cells enter S phase (24 h), and expression increases dramatically as cells progress through S phase. These data suggest that hDCC1 and hCTF18 are expressed only in actively dividing cells, consistent with their potential role in cohesion establishment in S phase. hCTF18, hCTF8, and hDCC1 Associate with RFC3—S. cerevisiae CTF18, CTF8, and DCC1 have been shown to interact with all four small RFC subunits. We next examined whether the human homologs would show similar interactions with RFC subunits. As shown in Fig. 4A, the p38 small subunit of replication factor C co-immunoprecipitated hCTF18, hCTF8, and hDCC1 and vice versa. Thus, it is likely that hCTF18, hCTF8, and hDCC1 form an alternative RFC complex similar to that observed in yeast. The hCTF18/hCTF8/hDCC1-containing Complex Co-immunoprecipitates with PCNA but Not the Rad9/Rad1/Hus1 Complex—Because the hCTF18/hCTF8/hDCC1-containing RFC complex is likely to function as a clamp loader complex, we sought to determine whether it interacts with any known DNA clamps. These include PCNA, which is involved in DNA synthesis, and the Rad9/Rad1/Hus1 complex, which is involved in the DNA damage response. We immunoprecipitated hCTF18, PCNA, and p38 RFC (as positive control) and Western blotted with αPCNA antibody. As shown in Fig. 4B (top panel), PCNA co-immunoprecipitated with p38 or hCTF18-C. As a negative control, rabbit IgG did not immunoprecipitate PCNA. Conversely, hCTF18 also co-immunoprecipitated with PCNA (Fig. 4B, bottom panel). However, only a very small amount of PCNA co-immunoprecipitated with hCTF18, leading us to speculate that PCNA may not be the clamp primarily associated with CTF18/CTF8/DCC1. Therefore, we performed similar experiment to look for an interaction with the Rad9/Rad1/Hus1 clamp. The Rad9/Rad1/Hus1 complex forms a PCNA-like clamp structure and is believed to be involved in DNA damage responses. We failed to detect any stable interaction between hCTF18/hCTF8/hDCC1 and the Rad9/Rad1/Hus1 complex (Fig. 4C and data not shown). As a positive control, we readily detected the interaction between Rad17 and the Rad9/Rad1/Hus1 complex (11Rauen M. Burtelow M.A. Dufault V.A. Karnitz L.M. J. Biol. Chem. 2000; 275: 29767-29771Abstract Full Text Full Text PDF PubMed Scopus (86) Google Scholar). In summary, these results show that hCTF18/hCTF8/hDCC1 selectively binds PCNA. This finding is supported by a recent paper that identified the putative hCTF18/RFC2–5 complex as a PCNA-binding partner through a proteomics approach (14Ohta S. Shiomi Y. Sugimoto K. Obuse C. Tsurimoto T. J. Biol. Chem. 2002; 277: 40362-40367Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). However, we cannot rule out that the hCTF18/hCTF8/hDCC1 complex may associate with another yet unidentified alternative clamp, which may be functionally redundant with PCNA under certain conditions. hCTF18, hCTF8, and hDCC1 Do Not Stably Associate with Cohesin Subunits—Because CTF18, CTF8, and DCC1 are thought to be involved in the establishment of sister chromatid cohesion in yeast, we next examined whether their human homologs interact with proteins in the cohesin complex. These include SMC1, SMC3, Pds5, and the two human SCC3 homologs SA1 and SA2. As shown in Fig. 4D, we failed to detect any stable interaction with members of the cohesin complex. Equivalent experiments in S. cerevisiae also failed to find a physical interaction of CTF18/CTF8/DCC1 with cohesion proteins (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar). It is possible that this alternative RFC complex does not directly interact with cohesion proteins or that this interaction may be very transient. hCTF18, hCTF8, and hDCC1 Are Predominantly Nuclear Proteins—We used immunofluorescence microscopy to determine the subcellular localization of hCTF18 and hDCC1. The proteins had a very similar staining pattern. They localized to nuclear heterochromatin in a diffuse manner and appeared less abundant in nucleoli. Their distribution did not appear to change over the cell cycle, except during mitosis, when hCTF18 and hDCC1 redistributed throughout the entire cell. S phase cells, identified by co-immunostaining with αPCNA (not shown), did not appear noticeably different in terms of hCTF18/hDCC1 staining when compared with non-S phase cells. hCTF18 Preferentially Associates with Chromatin during S Phase—Because we hypothesize that hCTF18 and hDCC1 may form part of an RFC-containing alternative clamp loader complex, we investigated whether its putative S phase function correlates with chromatin binding. Using a cell fractionation assay, we compared protein localization in HeLa cells arrested at different phases of the cell cycle. Whole cell lysate shows that hCTF18 protein level remains stable in actively cycling cells, regardless of the cell cycle stage. We collected mitotic cells by mitotic shake-off and S phase cells by synchronization using thymidine block or hydroxyurea treatment (see “Materials and Methods” for details). In mitotic cells, hCTF18 is present in the initial lysate, suggesting that the complex does not remain associated with chromatin during mitosis. However, only small amounts of hCTF18 are readily extractable from the nucleus during S phase. When S phase nuclei are treated with micrococcal nuclease to release chromatin-bound proteins, the majority of hCTF18 becomes solubilized. This result strongly suggests that hCTF18 associates with chromatin, not with the nuclear matrix, during S phase. To verify the integrity of the fractionation assay, we also blotted with αhOrc2p, one of the chromatin-associated proteins studied by the original authors using this fractionation assay (13Mendez J. Stillman B. Mol. Cell. Biol. 2000; 20: 8602-8612Crossref PubMed Scopus (755) Google Scholar). Finally, we reblotted with anti-β-actin as a loading control for whole cell and cytoplasmic fractions and with anti-CREB1 as a loading control for nuclear extracts (Fig. 5). The findings described above support the existence of a human alternative RFC complex composed of hCTF18, hCTF8, hDCC1, and RFC2–5. Because of its degree of evolutionary conservation, it is likely that the human complex is structurally and functionally similar to its S. cerevisiae homolog. We have shown that this complex binds PCNA but not the Rad9/Rad1/Hus1 complex, suggesting a certain degree of specificity. In addition, these novel human proteins strongly bind the p38 small subunit of replication factor C. The association of hCTF18/hCTF8/hDCC1 with both PCNA and p38 RFC provide strong evidence that these proteins may function as an alternative RFC-containing clamp loader complex. As expected, these proteins are nuclear and associate preferentially with chromatin during S phase. To date, this is the third known clamp loader complex to be described in humans. In the other two clamp loader systems (RFC1–5/PCNA and hRad17/RFC2–5/Rad9/Rad1/Hus1), there is a remarkable conservation of function between yeast and mammals. Further research will show whether this pattern holds true for the CTF18-containing complex. Despite many recent advances, the mechanism of sister cohesion establishment remains largely unknown. As reviewed by Carson and Christman (10Carson D.R. Christman M.F. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 8270-8275Crossref PubMed Scopus (65) Google Scholar), cohesion establishment appears to involve a polymerase switch from DNA polymerase δ to the newly described DNA polymerase σ (formerly polymerase κ). This switch is probably analogous to the polymerase α/polymerase δ switch mediated by RFC-PCNA during Okazaki fragment synthesis (12Waga S. Stillman B. Nature. 1994; 369: 207-212Crossref PubMed Scopus (497) Google Scholar). The novel clamp loader complex hCTF18/hCTF8/hDCC1/RFC (2Lee J.Y. Orr-Weaver T.L. Annu. Rev. Dev. Biol. 2001; 17: 753-757Crossref PubMed Scopus (139) Google Scholar, 3Nasmyth K. Annu. Rev. Genet. 2001; 35: 673-745Crossref PubMed Scopus (592) Google Scholar, 4Jallepalli P.V. Lengauer C. Nat. Rev. Cancer. 2001; 1: 109-117Crossref PubMed Scopus (339) Google Scholar, 5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) may replace RFC during replication over cohesin-bound DNA and associates with PCNA. However, it is not clear whether PCNA is the clamp preferentially bound by this novel complex or whether RFC(hCTF18/hCTF8/hDCC1) can recruit an additional clamp-like structure. Here, we have shown that there is no detectable association of hCTF18/hCTF8/hDCC1 with the hRad9/hRad1/hHus1 complex, a PCNA-like complex involved in the DNA damage response (15Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar). However, there are other proteins such as CTF7 that genetically interact with CTF18 and may form a PCNA-like clamp structure. The function of PCNA appears to overlap with that of CTF7, because overexpression of POL30 (PCNA) in yeast rescues the lethal phenotype of a temperature-sensitive mutant of CTF7 (16Skibbens R.V. Corson L.B. Koshland D. Hieter P. Genes Dev. 1999; 13: 307-319Crossref PubMed Scopus (385) Google Scholar). Mayer et al. (5Mayer M.L. Gygi S P. Aebersold R. Hieter P. Mol. Cell. 2001; 7: 959-970Abstract Full Text Full Text PDF PubMed Scopus (278) Google Scholar) noted that CTF18, CTF8, and DCC1 are excellent candidate genes for cancer predisposition, because their loss leads to an abnormal yet viable phenotype. If hCTF18/hCTF8/hDCC1 proves to be functionally as well as structurally equivalent to its S. cerevisiae homologs, it is likely that alterations of this complex may play a role in human disease. One of the earliest stages of tumor progression is the loss of genomic stability (17Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1997; 386: 623-627Crossref PubMed Scopus (1646) Google Scholar). Indeed, 80–90% of solid tumors are highly aneuploid, and this aneuploidy is thought to arise before frank transformation takes place. Therefore, genes that mediate chromosomal stability are potential early mutational targets in cancer. It remains to be determined whether the members of this novel RFC-containing complex are mutated in human malignancies or whether their loss can contribute to genomic instability. Further study of this novel replication factor C-containing complex will provide much needed insight into the poorly understood mechanism of sister chromatid cohesion establishment in humans. In addition, it may lead to a better understanding of the mechanism of aneuploidy during human carcinogenesis. We thank Drs. Scott Kaufmann and Ralf Janknecht for comments on the manuscript and Dr. Jun Qin for providing anti-SMC1 and anti-SMC3 antibodies." @default.
• W2046003114 created "2016-06-24" @default.
• W2046003114 creator A5029892501 @default.
• W2046003114 creator A5031335587 @default.
• W2046003114 creator A5044764908 @default.
• W2046003114 creator A5048801471 @default.
• W2046003114 date "2003-08-01" @default.
• W2046003114 modified "2023-10-03" @default.
• W2046003114 title "Cloning and Characterization of hCTF18, hCTF8, and hDCC1" @default.
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