SemOpenAlex |

SemOpenAlex

Matches in SemOpenAlex for { <https://semopenalex.org/work/W2053152019> ?p ?o ?g. }

Showing items 1 to 100 of ±108 with 100 items per page.

W2053152019 endingPage "29771" @default.
W2053152019 startingPage "29767" @default.
W2053152019 abstract "DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, andrad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA. DNA damage activates cell cycle checkpoints that prevent progression through the cell cycle. In yeast, the DNA damage checkpoint response is regulated by a series of genes that have mammalian homologs, including rad1, rad9, hus1, andrad17. On the basis of sequence homology, yeast and human Rad1, Rad9, and Hus1 protein homologs are predicted to structurally resemble the sliding clamp PCNA. Likewise, Rad17 homologs have extensive homology with replication factor C (RFC) subunits (p36, p37, p38, p40, and p140), which form a clamp loader for PCNA. These observations predict that Rad1, Hus1, and Rad9 might interact with Rad17 as a clamp-clamp loader pair during the DNA damage response. In this report, we demonstrate that endogenous human Rad17 (hRad17) interacts with the PCNA-related checkpoint proteins hRad1, hRad9, and hHus1. Mutational analysis of hRad1 and hRad17 demonstrates that this interaction has properties similar to the interaction between RFC and PCNA, a well characterized clamp-clamp loader pair. Moreover, we show that DNA damage affects the association of hRad17 with the clamp-like checkpoint proteins. Collectively, these data provide the first experimental evidence that hRad17 interacts with the PCNA-like proteins hRad1, hHus1, and hRad9 in manner similar to the interaction between RFC and PCNA. replication factor C polymerase chain reaction In response to DNA damage, eukaryotic cells block cell cycle progression in a process commonly known as the DNA damage-induced checkpoint response. Studies in genetically tractable yeast model systems have identified a large number of genes, dubbed checkpoint genes, that are essential for DNA damage-inducible checkpoint activation (reviewed in Refs. 1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar, 2Dasika G.K. Lin S.-C.J. Zhao S. Sung P. Tomkinson A. Lee E.Y.-H.P. Oncogene. 1999; 18: 7883-7899Crossref PubMed Scopus (349) Google Scholar, 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Epistasis and biochemical analyses in yeasts and humans have provisionally ordered the checkpoint proteins into a signaling pathway in which DNA damage relays activating signals through the phosphatidylinositol 3-kinase-related kinases, which include spRad3, scMec1, ATR, and ATM. The phosphatidylinositol 3-kinase-related kinases regulate activation of the serine-threonine protein kinases Chk1 and Chk2 (6Walworth N.C. Bernards R. Science. 1996; 271: 353-356Crossref PubMed Scopus (347) Google Scholar, 7Matsuoka S. Huang M.X. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 8Brown A.L. Lee C.H. Schwarz J.K. Mitiki N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (236) Google Scholar, 9Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (358) Google Scholar, 10Martinho R.G. Lindsay H.D. Flaggs G. DeMaggio A.J. Hoekstra M.F. Carr A.M. Bentley N.J. EMBO J. 1998; 17: 7239-7249Crossref PubMed Scopus (134) Google Scholar), which phosphorylate the cell-cycle phosphatase Cdc25 (7Matsuoka S. Huang M.X. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 9Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (358) Google Scholar, 11Zeng Y. Forbes K.C. Wu Z. Moreno S. Piwnica-Worms H. Enoch T. Nature. 1998; 395: 507-510Crossref PubMed Scopus (305) Google Scholar, 12Peng C.-Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar). Phosphorylation of Cdc25 inhibits its activity (13Furnari B. Blasina A. Boddy M.N. McGowan C.H. Russell P. Mol. Cell. Biol. 1999; 4: 833-845Crossref Scopus (177) Google Scholar, 14Blasina A. de Weyer I.V. Laus M.C. Luyten W.H. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and its accumulation in the nucleus (15Lopez-Girano A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar, 16Zeng Y. Piwnica-Worms H. Mol. Cell. Biol. 1999; 19: 7410-7419Crossref PubMed Scopus (133) Google Scholar), thereby preventing activation of the CyclinB-Cdc2 complex and blocking the G2/M transition after DNA damage.Studies in Schizosaccharomyces pombe and Saccharomyces cerevisiae demonstrated that the checkpoint proteins spRad1, spHus1, spRad9, and spRad17 (using S. pombe nomenclature) or their homologs are essential for DNA damage-activated checkpoint responses (reviewed in Refs. 1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar and 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Furthermore, these studies suggest that all four proteins act early in the DNA damage-induced signaling pathway. Sequence analyses provide a few clues regarding potential functions of these proteins. Yeast, human, and fly Rad1 exhibit sequence homology with Ustilago maydis Rec1 (17Bluyssen H.A.R. van Os R.I. Naus N.C. Jaspers I. Hoeijmakers J.H.J. de Klein A. Genomics. 1998; 54: 331-337Crossref PubMed Scopus (22) Google Scholar, 18Udell C.M. Lee S.K. Davey S. Nucleic Acids Res. 1998; 26: 3971-3976Crossref PubMed Scopus (35) Google Scholar, 19Parker A.E. Van de Weyer I. Laus M.C. Oostveen I. Yon J. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18332-18339Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Freire R. Murguia J.R. Tarsounas M. Lowndes N.F. Moens P.B. Jackson S.P. Genes Dev. 1998; 12: 2560-2573Crossref PubMed Scopus (102) Google Scholar, 21Marathi U.K. Dahlen M. Sunnerhagen P. Romero A.V. Ramagli L.R. Siciliano M.J. Li L. Legerski R.J. Genomics. 1998; 54: 344-347Crossref PubMed Scopus (21) Google Scholar, 22Dean F.B. Lian L. O'Donnell M. Genomics. 1998; 54: 424-436Crossref PubMed Scopus (48) Google Scholar, 23Long K.E. Sunnerhagen P. Subramani S. Gene. 1994; 148: 155-159Crossref PubMed Scopus (26) Google Scholar), a checkpoint protein and a 3′-5′exonuclease (24Thelen M.P. Onel K. Holloman W.K. J. Biol. Chem. 1994; 269: 747-754Abstract Full Text PDF PubMed Google Scholar), suggesting that Rad1 may also be a nuclease. However, a highly conserved DXE motif from nucleases is poorly conserved among mammalian Rad1 homologs (22Dean F.B. Lian L. O'Donnell M. Genomics. 1998; 54: 424-436Crossref PubMed Scopus (48) Google Scholar), and purified hRad1 has been variably reported to have nuclease activity (19Parker A.E. Van de Weyer I. Laus M.C. Oostveen I. Yon J. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18332-18339Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Freire R. Murguia J.R. Tarsounas M. Lowndes N.F. Moens P.B. Jackson S.P. Genes Dev. 1998; 12: 2560-2573Crossref PubMed Scopus (102) Google Scholar). Likewise, recombinant hRad9 has also been reported to possess nuclease activity (25Bessho T. Sancar A. J. Biol. Chem. 2000; 275: 7451-7454Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Recently, however, an alternative function for Rad1, Hus1, and Rad9 has been postulated. All three proteins exhibit sequence and possible structural homology with the sliding clamp protein PCNA (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 27Thelen M.P. Fidelis K. Cell. 1999; 96: 769-770Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Homotrimers of PCNA form a torodial structure that encircles DNA and tethers DNA polymerase δ to DNA during replication (reviewed in Refs.28Mossi R. Hubscher U. Eur. J. Biochem. 1998; 254: 209-216PubMed Google Scholar and 29Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (221) Google Scholar). This homology raises the possibility that Rad1, Rad9, and Hus1 may also form clamp-like structures that participate in the recognition or processing of damaged DNA. Consistent with this idea, Rad1, Rad9, and Hus1 have all been shown to interact with one another in cell lysates (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 30St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and by yeast two-hybrid analyses (30St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar, 32Hang H. Lieberman H.B. Genomics. 2000; 65: 24-33Crossref PubMed Scopus (56) Google Scholar). Additionally, we have recently shown that hRad1, hHus1, and hRad9 are converted to extraction-resistant nuclear foci following DNA damage (33Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar).Because clamp proteins encircle the DNA and are topologically linked with the DNA, they must be loaded onto the DNA by clamp loaders. The clamp loader for PCNA is replication factor C (RFC),1 a structure-specific heteropentameric complex (p36, p37, p38, p40, and p140) that recognizes primer-template junctions (28Mossi R. Hubscher U. Eur. J. Biochem. 1998; 254: 209-216PubMed Google Scholar, 29Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (221) Google Scholar). RFC, which is only fully functional as a pentamer (34Cai J. Yao N. Gibbs E. Finkelstein J. Phillips B. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11607-11612Crossref PubMed Scopus (44) Google Scholar, 35Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 36Ellison V. Stillman B. J. Biol. Chem. 1998; 273: 5979-5987Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 37Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 38Podust V.N. Tiwari N. Ott R. Fanning E. J. Biol. Chem. 1998; 273: 12935-12942Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), cracks the PCNA clamp and loads the clamp around the DNA in an ATP-dependent manner. If Rad1, Rad9, and Hus1 also form clamps, a clamp loader would likewise be required to load them onto the DNA. One potential clamp loader is the checkpoint protein Rad17, which exhibits significant homology with all five RFC subunits (39Bluyssen H.A.R. Naus N.C. van Os R.I. Jaspers I. Hoeijmakers J.H.J. de Klein A. Genomics. 1999; 55: 219-228Crossref PubMed Scopus (17) Google Scholar, 40Griffiths D.J.F. Barbet N.C. McCready S. Lehmann A.R. Carr A.M. EMBO J. 1995; 14: 5812-5823Crossref PubMed Scopus (178) Google Scholar, 41Li L. Peterson C.A. Kanter-Smoler G. Wei Y.-F. Ramagli L.S. Sunnerhagen P. Siciliano M.J. Legerski R.J. Oncogene. 1999; 18: 1689-1699Crossref PubMed Scopus (25) Google Scholar, 42Bao S. Chang M.-S. Auclair D. Sun Y. Wang Y. Wong W.-K. Zhang J. Liu Y. Qian X. Sutherland R. Magi-Galluzi C. Weisberg E. Cheng E.Y.S. Hao L. Sasaki H. Campbell M.S. Kraeft S.-K. Lod M. Lo K.-M. Chen L.B. Cancer Res. 1999; 59: 2023-2028PubMed Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The homology appears related to function. TheS. cerevisiae homolog of Rad17 (scRad24) interacts with four of the five RFC subunits, forming a distinct multimeric structure in which Rad17 (scRad24) replaces the large RFC subunit in the complex (44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar).Taken together, these observations suggest a tentative model in which a Rad17 clamp loader may interact with the Rad1, Hus1, and Rad9 clamp-like proteins during recognition or processing of DNA damage. This model predicts that Rad17 should interact with Rad1, Hus1, or Rad9. To date, this model has been largely untested. Although an interaction between Rad1 and Rad17 homologs has been documented by yeast two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), several groups have reported that endogenous Rad17 does not interact with Rad1 (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 45Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar). We now report that hRad17 does indeed interact with all three PCNA-like proteins, hRad1, hRad9, and hHus1, in cell lysates. Furthermore, by mutational analysis, we demonstrate that the interaction between hRad17 and hRad1 has biochemical features similar to the RFC-PCNA interaction. Finally, we show that DNA damage alters the interaction of hRad17 with hHus1, hRad1, and hRad9.DISCUSSIONThe checkpoint proteins Rad1, Hus1, Rad9, and Rad17 are required for early events in the activation of the DNA damage checkpoint-signaling pathway (1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar, 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Initially, there were few clues regarding their functions. Recently, however, Rad1, Hus1, and Rad9 have been predicted to be PCNA-like clamp proteins (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 27Thelen M.P. Fidelis K. Cell. 1999; 96: 769-770Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and S. cerevisiae Rad17 (scRad24), which is homologous with RFC subunits, was found to interact with four of the five RFC subunits, suggesting that it may be a clamp loader (44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Collectively, these observations suggest that DNA damage-induced checkpoint activation requires a clamp and a clamp loader. One prediction from this model is that the components of the clamp (Rad1) and clamp loader (Rad17) interact. Although several groups showed that Rad17 and Rad1 homologs interacted in yeast two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), initially, we and others could not demonstrate an interaction between endogenous Rad17 and Rad1 homologs (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 45Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar). Here, for the first time, we demonstrate that endogenous hRad17 and hRad1 interact, and we also show that hRad17 interacts with hRad9 and hHus1, two other PCNA-like checkpoint proteins. One possible organization for this complex is with hRad1, hHus1, and hRad9 forming a clamp-like complex, with hRad1 linking an hRad1-hHus1-hRad9 complex to hRad17 (Fig.5). This idea is supported by the finding that yeast and human Rad17 interacts with Rad1 but not Rad9 or Hus1 in two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and by our present observation that the hRad1 M3 mutant disrupts the interaction with hRad17 but not hRad9 and hHus1.Additionally, our studies with mutant hRad17 and hRad1 suggest that the interactions between hRad17 and hRad1 may be similar to those between RFC and PCNA. First, mutations that disrupt the nucleotide-binding domain of hRad17 prevent interaction with hRad1 but not RFC p38, suggesting that nucleotide binding is important for stable association with hRad1. A similar requirement for nucleotide binding has been noted for RFC-PCNA interactions: in the presence of ATP and Mg2+, RFC interacted more tightly with PCNA (35Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Second, the hRad1 M3 mutation selectively disrupted interaction between hRad1 and hRad17. The analogous region in PCNA is also required for productive interactions between PCNA and RFC (48Zhang G. Gibbs E. Kelman Z. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1869-1874Crossref PubMed Scopus (72) Google Scholar), suggesting that parallel regions of hRad1 and PCNA participate in interactions with clamp loaders.Several lines of investigation now suggest a provocative model regarding the roles of hRad1, hHus1, hRad9, and hRad17 in DNA damage-activated responses. In this model, an hRad17-containing clamp would recognize structural alterations induced by DNA damage (Fig. 5), much as classical RFC complexes recognize primer-template junctions. The hRad17-containing clamp loader would then load hRad1, hHus1, and hRad9 clamps onto DNA, thus converting them from readily extractable nuclear proteins to extraction-resistant nuclear foci (33Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, the results presented in Fig. 4 demonstrate that even after DNA damage, hRad17 does not stably associate with nuclear elements, suggesting that hRad17 may interact only transiently with DNA. Once hRad9, hHus1, and hRad1 are loaded onto DNA, they may then recruit proteins that participate in DNA processing or activation of the downstream checkpoint signaling machinery. In response to DNA damage, eukaryotic cells block cell cycle progression in a process commonly known as the DNA damage-induced checkpoint response. Studies in genetically tractable yeast model systems have identified a large number of genes, dubbed checkpoint genes, that are essential for DNA damage-inducible checkpoint activation (reviewed in Refs. 1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar, 2Dasika G.K. Lin S.-C.J. Zhao S. Sung P. Tomkinson A. Lee E.Y.-H.P. Oncogene. 1999; 18: 7883-7899Crossref PubMed Scopus (349) Google Scholar, 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Epistasis and biochemical analyses in yeasts and humans have provisionally ordered the checkpoint proteins into a signaling pathway in which DNA damage relays activating signals through the phosphatidylinositol 3-kinase-related kinases, which include spRad3, scMec1, ATR, and ATM. The phosphatidylinositol 3-kinase-related kinases regulate activation of the serine-threonine protein kinases Chk1 and Chk2 (6Walworth N.C. Bernards R. Science. 1996; 271: 353-356Crossref PubMed Scopus (347) Google Scholar, 7Matsuoka S. Huang M.X. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 8Brown A.L. Lee C.H. Schwarz J.K. Mitiki N. Piwnica-Worms H. Chung J.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 3745-3750Crossref PubMed Scopus (236) Google Scholar, 9Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (358) Google Scholar, 10Martinho R.G. Lindsay H.D. Flaggs G. DeMaggio A.J. Hoekstra M.F. Carr A.M. Bentley N.J. EMBO J. 1998; 17: 7239-7249Crossref PubMed Scopus (134) Google Scholar), which phosphorylate the cell-cycle phosphatase Cdc25 (7Matsuoka S. Huang M.X. Elledge S.J. Science. 1998; 282: 1893-1897Crossref PubMed Scopus (1078) Google Scholar, 9Chaturvedi P. Eng W.K. Zhu Y. Mattern M.R. Mishra R. Hurle M.R. Zhang X. Annan R.S. Lu Q. Faucette L.F. Scott G.F. Li X. Carr S.A. Johnson R.K. Winkler J.D. Zhou B.B. Oncogene. 1999; 18: 4047-4054Crossref PubMed Scopus (358) Google Scholar, 11Zeng Y. Forbes K.C. Wu Z. Moreno S. Piwnica-Worms H. Enoch T. Nature. 1998; 395: 507-510Crossref PubMed Scopus (305) Google Scholar, 12Peng C.-Y. Graves P.R. Thoma R.S. Wu Z. Shaw A.S. Piwnica-Worms H. Science. 1997; 277: 1501-1505Crossref PubMed Scopus (1178) Google Scholar). Phosphorylation of Cdc25 inhibits its activity (13Furnari B. Blasina A. Boddy M.N. McGowan C.H. Russell P. Mol. Cell. Biol. 1999; 4: 833-845Crossref Scopus (177) Google Scholar, 14Blasina A. de Weyer I.V. Laus M.C. Luyten W.H. Parker A.E. McGowan C.H. Curr. Biol. 1999; 9: 1-10Abstract Full Text Full Text PDF PubMed Scopus (242) Google Scholar) and its accumulation in the nucleus (15Lopez-Girano A. Furnari B. Mondesert O. Russell P. Nature. 1999; 397: 172-175Crossref PubMed Scopus (501) Google Scholar, 16Zeng Y. Piwnica-Worms H. Mol. Cell. Biol. 1999; 19: 7410-7419Crossref PubMed Scopus (133) Google Scholar), thereby preventing activation of the CyclinB-Cdc2 complex and blocking the G2/M transition after DNA damage. Studies in Schizosaccharomyces pombe and Saccharomyces cerevisiae demonstrated that the checkpoint proteins spRad1, spHus1, spRad9, and spRad17 (using S. pombe nomenclature) or their homologs are essential for DNA damage-activated checkpoint responses (reviewed in Refs. 1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar and 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Furthermore, these studies suggest that all four proteins act early in the DNA damage-induced signaling pathway. Sequence analyses provide a few clues regarding potential functions of these proteins. Yeast, human, and fly Rad1 exhibit sequence homology with Ustilago maydis Rec1 (17Bluyssen H.A.R. van Os R.I. Naus N.C. Jaspers I. Hoeijmakers J.H.J. de Klein A. Genomics. 1998; 54: 331-337Crossref PubMed Scopus (22) Google Scholar, 18Udell C.M. Lee S.K. Davey S. Nucleic Acids Res. 1998; 26: 3971-3976Crossref PubMed Scopus (35) Google Scholar, 19Parker A.E. Van de Weyer I. Laus M.C. Oostveen I. Yon J. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18332-18339Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Freire R. Murguia J.R. Tarsounas M. Lowndes N.F. Moens P.B. Jackson S.P. Genes Dev. 1998; 12: 2560-2573Crossref PubMed Scopus (102) Google Scholar, 21Marathi U.K. Dahlen M. Sunnerhagen P. Romero A.V. Ramagli L.R. Siciliano M.J. Li L. Legerski R.J. Genomics. 1998; 54: 344-347Crossref PubMed Scopus (21) Google Scholar, 22Dean F.B. Lian L. O'Donnell M. Genomics. 1998; 54: 424-436Crossref PubMed Scopus (48) Google Scholar, 23Long K.E. Sunnerhagen P. Subramani S. Gene. 1994; 148: 155-159Crossref PubMed Scopus (26) Google Scholar), a checkpoint protein and a 3′-5′exonuclease (24Thelen M.P. Onel K. Holloman W.K. J. Biol. Chem. 1994; 269: 747-754Abstract Full Text PDF PubMed Google Scholar), suggesting that Rad1 may also be a nuclease. However, a highly conserved DXE motif from nucleases is poorly conserved among mammalian Rad1 homologs (22Dean F.B. Lian L. O'Donnell M. Genomics. 1998; 54: 424-436Crossref PubMed Scopus (48) Google Scholar), and purified hRad1 has been variably reported to have nuclease activity (19Parker A.E. Van de Weyer I. Laus M.C. Oostveen I. Yon J. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18332-18339Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar, 20Freire R. Murguia J.R. Tarsounas M. Lowndes N.F. Moens P.B. Jackson S.P. Genes Dev. 1998; 12: 2560-2573Crossref PubMed Scopus (102) Google Scholar). Likewise, recombinant hRad9 has also been reported to possess nuclease activity (25Bessho T. Sancar A. J. Biol. Chem. 2000; 275: 7451-7454Abstract Full Text Full Text PDF PubMed Scopus (69) Google Scholar). Recently, however, an alternative function for Rad1, Hus1, and Rad9 has been postulated. All three proteins exhibit sequence and possible structural homology with the sliding clamp protein PCNA (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 27Thelen M.P. Fidelis K. Cell. 1999; 96: 769-770Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar). Homotrimers of PCNA form a torodial structure that encircles DNA and tethers DNA polymerase δ to DNA during replication (reviewed in Refs.28Mossi R. Hubscher U. Eur. J. Biochem. 1998; 254: 209-216PubMed Google Scholar and 29Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (221) Google Scholar). This homology raises the possibility that Rad1, Rad9, and Hus1 may also form clamp-like structures that participate in the recognition or processing of damaged DNA. Consistent with this idea, Rad1, Rad9, and Hus1 have all been shown to interact with one another in cell lysates (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 30St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar) and by yeast two-hybrid analyses (30St. Onge R.P. Udell C.M. Casselman R. Davey S. Mol. Biol. Cell. 1999; 10: 1985-1995Crossref PubMed Scopus (129) Google Scholar, 32Hang H. Lieberman H.B. Genomics. 2000; 65: 24-33Crossref PubMed Scopus (56) Google Scholar). Additionally, we have recently shown that hRad1, hHus1, and hRad9 are converted to extraction-resistant nuclear foci following DNA damage (33Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Because clamp proteins encircle the DNA and are topologically linked with the DNA, they must be loaded onto the DNA by clamp loaders. The clamp loader for PCNA is replication factor C (RFC),1 a structure-specific heteropentameric complex (p36, p37, p38, p40, and p140) that recognizes primer-template junctions (28Mossi R. Hubscher U. Eur. J. Biochem. 1998; 254: 209-216PubMed Google Scholar, 29Stillman B. Cell. 1994; 78: 725-728Abstract Full Text PDF PubMed Scopus (221) Google Scholar). RFC, which is only fully functional as a pentamer (34Cai J. Yao N. Gibbs E. Finkelstein J. Phillips B. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 11607-11612Crossref PubMed Scopus (44) Google Scholar, 35Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 36Ellison V. Stillman B. J. Biol. Chem. 1998; 273: 5979-5987Abstract Full Text Full Text PDF PubMed Scopus (49) Google Scholar, 37Podust V.N. Fanning E. J. Biol. Chem. 1997; 272: 6303-6310Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar, 38Podust V.N. Tiwari N. Ott R. Fanning E. J. Biol. Chem. 1998; 273: 12935-12942Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar), cracks the PCNA clamp and loads the clamp around the DNA in an ATP-dependent manner. If Rad1, Rad9, and Hus1 also form clamps, a clamp loader would likewise be required to load them onto the DNA. One potential clamp loader is the checkpoint protein Rad17, which exhibits significant homology with all five RFC subunits (39Bluyssen H.A.R. Naus N.C. van Os R.I. Jaspers I. Hoeijmakers J.H.J. de Klein A. Genomics. 1999; 55: 219-228Crossref PubMed Scopus (17) Google Scholar, 40Griffiths D.J.F. Barbet N.C. McCready S. Lehmann A.R. Carr A.M. EMBO J. 1995; 14: 5812-5823Crossref PubMed Scopus (178) Google Scholar, 41Li L. Peterson C.A. Kanter-Smoler G. Wei Y.-F. Ramagli L.S. Sunnerhagen P. Siciliano M.J. Legerski R.J. Oncogene. 1999; 18: 1689-1699Crossref PubMed Scopus (25) Google Scholar, 42Bao S. Chang M.-S. Auclair D. Sun Y. Wang Y. Wong W.-K. Zhang J. Liu Y. Qian X. Sutherland R. Magi-Galluzi C. Weisberg E. Cheng E.Y.S. Hao L. Sasaki H. Campbell M.S. Kraeft S.-K. Lod M. Lo K.-M. Chen L.B. Cancer Res. 1999; 59: 2023-2028PubMed Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar). The homology appears related to function. TheS. cerevisiae homolog of Rad17 (scRad24) interacts with four of the five RFC subunits, forming a distinct multimeric structure in which Rad17 (scRad24) replaces the large RFC subunit in the complex (44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Taken together, these observations suggest a tentative model in which a Rad17 clamp loader may interact with the Rad1, Hus1, and Rad9 clamp-like proteins during recognition or processing of DNA damage. This model predicts that Rad17 should interact with Rad1, Hus1, or Rad9. To date, this model has been largely untested. Although an interaction between Rad1 and Rad17 homologs has been documented by yeast two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), several groups have reported that endogenous Rad17 does not interact with Rad1 (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 45Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar). We now report that hRad17 does indeed interact with all three PCNA-like proteins, hRad1, hRad9, and hHus1, in cell lysates. Furthermore, by mutational analysis, we demonstrate that the interaction between hRad17 and hRad1 has biochemical features similar to the RFC-PCNA interaction. Finally, we show that DNA damage alters the interaction of hRad17 with hHus1, hRad1, and hRad9. DISCUSSIONThe checkpoint proteins Rad1, Hus1, Rad9, and Rad17 are required for early events in the activation of the DNA damage checkpoint-signaling pathway (1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar, 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Initially, there were few clues regarding their functions. Recently, however, Rad1, Hus1, and Rad9 have been predicted to be PCNA-like clamp proteins (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 27Thelen M.P. Fidelis K. Cell. 1999; 96: 769-770Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and S. cerevisiae Rad17 (scRad24), which is homologous with RFC subunits, was found to interact with four of the five RFC subunits, suggesting that it may be a clamp loader (44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Collectively, these observations suggest that DNA damage-induced checkpoint activation requires a clamp and a clamp loader. One prediction from this model is that the components of the clamp (Rad1) and clamp loader (Rad17) interact. Although several groups showed that Rad17 and Rad1 homologs interacted in yeast two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), initially, we and others could not demonstrate an interaction between endogenous Rad17 and Rad1 homologs (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 45Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar). Here, for the first time, we demonstrate that endogenous hRad17 and hRad1 interact, and we also show that hRad17 interacts with hRad9 and hHus1, two other PCNA-like checkpoint proteins. One possible organization for this complex is with hRad1, hHus1, and hRad9 forming a clamp-like complex, with hRad1 linking an hRad1-hHus1-hRad9 complex to hRad17 (Fig.5). This idea is supported by the finding that yeast and human Rad17 interacts with Rad1 but not Rad9 or Hus1 in two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and by our present observation that the hRad1 M3 mutant disrupts the interaction with hRad17 but not hRad9 and hHus1.Additionally, our studies with mutant hRad17 and hRad1 suggest that the interactions between hRad17 and hRad1 may be similar to those between RFC and PCNA. First, mutations that disrupt the nucleotide-binding domain of hRad17 prevent interaction with hRad1 but not RFC p38, suggesting that nucleotide binding is important for stable association with hRad1. A similar requirement for nucleotide binding has been noted for RFC-PCNA interactions: in the presence of ATP and Mg2+, RFC interacted more tightly with PCNA (35Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Second, the hRad1 M3 mutation selectively disrupted interaction between hRad1 and hRad17. The analogous region in PCNA is also required for productive interactions between PCNA and RFC (48Zhang G. Gibbs E. Kelman Z. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1869-1874Crossref PubMed Scopus (72) Google Scholar), suggesting that parallel regions of hRad1 and PCNA participate in interactions with clamp loaders.Several lines of investigation now suggest a provocative model regarding the roles of hRad1, hHus1, hRad9, and hRad17 in DNA damage-activated responses. In this model, an hRad17-containing clamp would recognize structural alterations induced by DNA damage (Fig. 5), much as classical RFC complexes recognize primer-template junctions. The hRad17-containing clamp loader would then load hRad1, hHus1, and hRad9 clamps onto DNA, thus converting them from readily extractable nuclear proteins to extraction-resistant nuclear foci (33Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, the results presented in Fig. 4 demonstrate that even after DNA damage, hRad17 does not stably associate with nuclear elements, suggesting that hRad17 may interact only transiently with DNA. Once hRad9, hHus1, and hRad1 are loaded onto DNA, they may then recruit proteins that participate in DNA processing or activation of the downstream checkpoint signaling machinery. The checkpoint proteins Rad1, Hus1, Rad9, and Rad17 are required for early events in the activation of the DNA damage checkpoint-signaling pathway (1Longhese M.P. Foiani M. Muzi-Falconi M. Lucchini G. Plevani P. EMBO J. 1998; 17: 5525-5528Crossref PubMed Scopus (141) Google Scholar, 3Caspari T. Carr A.M. Biochimie. 1999; 81: 173-181Crossref PubMed Scopus (79) Google Scholar, 4Lowndes N.F. Murguia J.R. Curr. Opin. Genet. Dev. 2000; 10: 17-25Crossref PubMed Scopus (234) Google Scholar, 5Weinert T. Cell. 1998; 94: 555-558Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar). Initially, there were few clues regarding their functions. Recently, however, Rad1, Hus1, and Rad9 have been predicted to be PCNA-like clamp proteins (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 27Thelen M.P. Fidelis K. Cell. 1999; 96: 769-770Abstract Full Text Full Text PDF PubMed Scopus (130) Google Scholar), and S. cerevisiae Rad17 (scRad24), which is homologous with RFC subunits, was found to interact with four of the five RFC subunits, suggesting that it may be a clamp loader (44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar). Collectively, these observations suggest that DNA damage-induced checkpoint activation requires a clamp and a clamp loader. One prediction from this model is that the components of the clamp (Rad1) and clamp loader (Rad17) interact. Although several groups showed that Rad17 and Rad1 homologs interacted in yeast two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar), initially, we and others could not demonstrate an interaction between endogenous Rad17 and Rad1 homologs (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 31Volkmer E. Karnitz L.M. J. Biol. Chem. 1999; 274: 567-570Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 44Green C.M. Erdjument-Bromage H. Tempst P. Lowndes N.F. Curr. Biol. 2000; 10: 39-42Abstract Full Text Full Text PDF PubMed Scopus (187) Google Scholar, 45Edwards R.J. Bentley N.J. Carr A.M. Nat. Cell Biol. 1999; 1: 393-398Crossref PubMed Scopus (172) Google Scholar). Here, for the first time, we demonstrate that endogenous hRad17 and hRad1 interact, and we also show that hRad17 interacts with hRad9 and hHus1, two other PCNA-like checkpoint proteins. One possible organization for this complex is with hRad1, hHus1, and hRad9 forming a clamp-like complex, with hRad1 linking an hRad1-hHus1-hRad9 complex to hRad17 (Fig.5). This idea is supported by the finding that yeast and human Rad17 interacts with Rad1 but not Rad9 or Hus1 in two-hybrid analyses (26Caspari T. Dahlen M. Kanter-Smoler G. Lindsay H.D. Hofmann K. Papadimitriou K. Sunnerhagen P. Carr A.M. Mol. Cell. Biol. 2000; 74: 1254-1262Crossref Scopus (205) Google Scholar, 43Parker A.E. Van de Weyer I. Laus M.C. Verhasselt P. Luyten W.H.M.L. J. Biol. Chem. 1998; 273: 18340-18346Abstract Full Text Full Text PDF PubMed Scopus (72) Google Scholar) and by our present observation that the hRad1 M3 mutant disrupts the interaction with hRad17 but not hRad9 and hHus1. Additionally, our studies with mutant hRad17 and hRad1 suggest that the interactions between hRad17 and hRad1 may be similar to those between RFC and PCNA. First, mutations that disrupt the nucleotide-binding domain of hRad17 prevent interaction with hRad1 but not RFC p38, suggesting that nucleotide binding is important for stable association with hRad1. A similar requirement for nucleotide binding has been noted for RFC-PCNA interactions: in the presence of ATP and Mg2+, RFC interacted more tightly with PCNA (35Gerik K.J. Gary S.L. Burgers P.M.J. J. Biol. Chem. 1997; 272: 1256-1262Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar). Second, the hRad1 M3 mutation selectively disrupted interaction between hRad1 and hRad17. The analogous region in PCNA is also required for productive interactions between PCNA and RFC (48Zhang G. Gibbs E. Kelman Z. O'Donnell M. Hurwitz J. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 1869-1874Crossref PubMed Scopus (72) Google Scholar), suggesting that parallel regions of hRad1 and PCNA participate in interactions with clamp loaders. Several lines of investigation now suggest a provocative model regarding the roles of hRad1, hHus1, hRad9, and hRad17 in DNA damage-activated responses. In this model, an hRad17-containing clamp would recognize structural alterations induced by DNA damage (Fig. 5), much as classical RFC complexes recognize primer-template junctions. The hRad17-containing clamp loader would then load hRad1, hHus1, and hRad9 clamps onto DNA, thus converting them from readily extractable nuclear proteins to extraction-resistant nuclear foci (33Burtelow M.A. Kaufmann S.H. Karnitz L.M. J. Biol. Chem. 2000; 275: 26343-26348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). However, the results presented in Fig. 4 demonstrate that even after DNA damage, hRad17 does not stably associate with nuclear elements, suggesting that hRad17 may interact only transiently with DNA. Once hRad9, hHus1, and hRad1 are loaded onto DNA, they may then recruit proteins that participate in DNA processing or activation of the downstream checkpoint signaling machinery. We thank Drs. Scott Kaufmann and Junjie Chen for critical reading of the manuscript. We thank Dr. Vladimir Podust for RFC p38 cDNA and Wanda Rhodes for excellent manuscript preparation." @default.
W2053152019 created "2016-06-24" @default.
W2053152019 creator A5019153456 @default.
W2053152019 creator A5043726924 @default.
W2053152019 creator A5048801471 @default.
W2053152019 creator A5065579408 @default.
W2053152019 date "2000-09-01" @default.
W2053152019 modified "2023-10-17" @default.
W2053152019 title "The Human Checkpoint Protein hRad17 Interacts with the PCNA-like Proteins hRad1, hHus1, and hRad9" @default.
W2053152019 cites W1504970629 @default.
W2053152019 cites W1520947267 @default.
W2053152019 cites W1621777818 @default.
W2053152019 cites W1677118171 @default.
W2053152019 cites W1968687444 @default.
W2053152019 cites W1974441944 @default.
W2053152019 cites W1975098427 @default.
W2053152019 cites W1976655581 @default.
W2053152019 cites W1984415680 @default.
W2053152019 cites W1985859625 @default.
W2053152019 cites W1988840026 @default.
W2053152019 cites W1988876917 @default.
W2053152019 cites W1994848650 @default.
W2053152019 cites W1994909028 @default.
W2053152019 cites W2011962432 @default.
W2053152019 cites W2014722055 @default.
W2053152019 cites W2018705198 @default.
W2053152019 cites W2019068612 @default.
W2053152019 cites W2033126648 @default.
W2053152019 cites W2043136696 @default.
W2053152019 cites W2052801384 @default.
W2053152019 cites W2054311041 @default.
W2053152019 cites W2055896601 @default.
W2053152019 cites W2060020768 @default.
W2053152019 cites W2075571931 @default.
W2053152019 cites W2076314401 @default.
W2053152019 cites W2080140141 @default.
W2053152019 cites W2082747505 @default.
W2053152019 cites W2082941148 @default.
W2053152019 cites W2090002023 @default.
W2053152019 cites W2106049510 @default.
W2053152019 cites W2109482063 @default.
W2053152019 cites W2111975122 @default.
W2053152019 cites W2127886165 @default.
W2053152019 cites W2128994729 @default.
W2053152019 cites W2140730373 @default.
W2053152019 cites W2147971204 @default.
W2053152019 cites W2154052847 @default.
W2053152019 cites W2159040452 @default.
W2053152019 cites W2160954038 @default.
W2053152019 cites W2167391339 @default.
W2053152019 cites W2170264107 @default.
W2053152019 cites W2178835627 @default.
W2053152019 cites W2327471215 @default.
W2053152019 cites W4241839321 @default.
W2053152019 doi "https://doi.org/10.1074/jbc.m005782200" @default.
W2053152019 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/10884395" @default.
W2053152019 hasPublicationYear "2000" @default.
W2053152019 type Work @default.
W2053152019 sameAs 2053152019 @default.
W2053152019 citedByCount "108" @default.
W2053152019 countsByYear W20531520192012 @default.
W2053152019 countsByYear W20531520192013 @default.
W2053152019 countsByYear W20531520192014 @default.
W2053152019 countsByYear W20531520192015 @default.
W2053152019 countsByYear W20531520192016 @default.
W2053152019 countsByYear W20531520192017 @default.
W2053152019 countsByYear W20531520192019 @default.
W2053152019 countsByYear W20531520192021 @default.
W2053152019 countsByYear W20531520192023 @default.
W2053152019 crossrefType "journal-article" @default.
W2053152019 hasAuthorship W2053152019A5019153456 @default.
W2053152019 hasAuthorship W2053152019A5043726924 @default.
W2053152019 hasAuthorship W2053152019A5048801471 @default.
W2053152019 hasAuthorship W2053152019A5065579408 @default.
W2053152019 hasBestOaLocation W20531520191 @default.
W2053152019 hasConcept C185592680 @default.
W2053152019 hasConcept C34688535 @default.
W2053152019 hasConcept C552990157 @default.
W2053152019 hasConcept C55493867 @default.
W2053152019 hasConcept C86803240 @default.
W2053152019 hasConcept C95444343 @default.
W2053152019 hasConceptScore W2053152019C185592680 @default.
W2053152019 hasConceptScore W2053152019C34688535 @default.
W2053152019 hasConceptScore W2053152019C552990157 @default.
W2053152019 hasConceptScore W2053152019C55493867 @default.
W2053152019 hasConceptScore W2053152019C86803240 @default.
W2053152019 hasConceptScore W2053152019C95444343 @default.
W2053152019 hasIssue "38" @default.
W2053152019 hasLocation W20531520191 @default.
W2053152019 hasOpenAccess W2053152019 @default.
W2053152019 hasPrimaryLocation W20531520191 @default.
W2053152019 hasRelatedWork W1531601525 @default.
W2053152019 hasRelatedWork W2319480705 @default.
W2053152019 hasRelatedWork W2384464875 @default.
W2053152019 hasRelatedWork W2606230654 @default.
W2053152019 hasRelatedWork W2607424097 @default.
W2053152019 hasRelatedWork W2748952813 @default.
W2053152019 hasRelatedWork W2899084033 @default.