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An Analysis of CAF-1-interacting Proteins Reveals Dynamic and Direct Interactions with the KU Complex and 14-3-3 Proteins

2011; Elsevier BV; Volume: 286; Issue: 12 Linguagem: Inglês

10.1074/jbc.m110.217075

ISSN

1083-351X

Autores

Maarten Hoek, Michael P. Myers, Bruce Stillman,

Tópico(s)

DNA Repair Mechanisms

Resumo

CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly. CAF-1 is essential in human cells for the de novo deposition of histones H3 and H4 at the DNA replication fork. Depletion of CAF-1 from various cell lines causes replication fork arrest, activation of the intra-S phase checkpoint, and global defects in chromatin structure. CAF-1 is also involved in coordinating inheritance of states of gene expression and in chromatin assembly following DNA repair. In this study, we generated cell lines expressing RNAi-resistant versions of CAF-1 and showed that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 was deficient in proliferating cell nuclear antigen (PCNA) binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding had no effect on the recruitment of CAF-1 to chromatin after DNA damage or to resistance to DNA-damaging agents. Tandem affinity purification of CAF-1-interacting proteins under mild conditions revealed that CAF-1 was directly associated with the KU70/80 complex, part of the DNA-dependent protein kinase, and the phosphoserine/threonine-binding protein 14-3-3 ζ. CAF-1 was a substrate for DNA-dependent protein kinase, and the 14-3-3 interaction in vitro is dependent on DNA-dependent protein kinase phosphorylation. These results highlight that CAF-1 has prominent interactions with the DNA repair machinery but that the N terminus is dispensable for the role of CAF-1 in DNA replication- and repair-coupled chromatin assembly. IntroductionDNA in eukaryotic cells is packaged into nucleosomes that form higher order chromatin. During DNA replication, histones H3 and H4 that form the core of the nucleosome are randomly partitioned to the daughter strands, and new histones are deposited de novo to maintain histone density in the wake of the replication fork (1Verreault A. Genes Dev. 2000; 14: 1430-1438PubMed Google Scholar). Although many histone chaperones exist in human cells, the primary DNA replication-coupled histone deposition factor is CAF-1 as determined from biochemical purification experiments (2Smith S. Stillman B. Cell. 1989; 58: 15-25Abstract Full Text PDF PubMed Scopus (513) Google Scholar). Depletion of CAF-1 from human cells has been shown to lead to defects in DNA replication fork progression, activation of the intra-S phase checkpoint, and global defects in chromatin structure (3Hoek M. Stillman B. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12183-12188Crossref PubMed Scopus (189) Google Scholar4Nabatiyan A. Szüts D. Krude T. Mol. Cell. Biol. 2006; 26: 1839-1849Crossref PubMed Scopus (39) Google Scholar5Ye X. Franco A.A. Santos H. Nelson D.M. Kaufman P.D. Adams P.D. Mol. Cell. 2003; 11: 341-351Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar).CAF-1 is a heterotrimeric complex consisting of 150-, 60-, and 48-kDa subunits that binds to histones H3 and H4 (6Kaufman P.D. Kobayashi R. Kessler N. Stillman B. Cell. 1995; 81: 1105-1114Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 7Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Cell. 1996; 87: 95-104Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). The p60 and p48 subunits consist largely of WD40 repeats that play a role in histone binding, and p48 is known to be a member of numerous H3/H4 binding complexes such as HAT1 and HDAC1 (8Ahmad A. Takami Y. Nakayama T. J. Biol. Chem. 1999; 274: 16646-16653Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar9Tie F. Furuyama T. Prasad-Sinha J. Jane E. Harte P.J. Development. 2001; 128: 275-286Crossref PubMed Google Scholar10Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1998; 8: 96-108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). CAF-1 p60 also interacts with the histone chaperones ASF1A and ASF1B through their B-domain, which resides outside of the WD40 repeat region (11Tang Y. Poustovoitov M.V. Zhao K. Garfinkel M. Canutescu A. Dunbrack R. Adams P.D. Marmorstein R. Nat. Struct. Mol. Biol. 2006; 13: 921-929Crossref PubMed Scopus (131) Google Scholar). The p150 subunit has been better characterized and contains the regions that mediate interactions with proliferating cell nuclear antigen (PCNA), 4The abbreviations used are: PCNA, proliferating cell nuclear antigen; DNA-PK, DNA-dependent protein kinase; MMS, methane methylsulfonate; DNA-PKCS, DNA-dependent protein kinase catalytic subunit; LC-MS/MS, liquid chromatography-tandem mass spectrometry; CDK, cyclin-dependent kinase. HP1, MBD1, BLM, and the CAF-1 p60 subunit. The PCNA mediates the connection between CAF-1 and the DNA replication fork (12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar13Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar14Zhang Z. Shibahara K. Stillman B. Nature. 2000; 408: 221-225Crossref PubMed Scopus (384) Google Scholar). There are two PCNA binding sites that have been mapped in the p150 subunit, but CAF-1 is localized to replication forks through a conserved PCNA binding region motif called PIP2 in the middle of the p150 open reading frame (5Ye X. Franco A.A. Santos H. Nelson D.M. Kaufman P.D. Adams P.D. Mol. Cell. 2003; 11: 341-351Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar, 15Krawitz D.C. Kama T. Kaufman P.D. Mol. Cell. Biol. 2002; 22: 614-625Crossref PubMed Scopus (135) Google Scholar). The N terminus of p150, containing a second PCNA binding region as well as the HP1-interacting region, is dispensable for DNA replication-coupled CAF-1 chromatin assembly activity in vitro and for localization to replication foci in vivo (12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar, 16Moggs J.G. Grandi P. Quivy J.P. Jónsson Z.O. Hübscher U. Becker P.B. Almouzni G. Mol. Cell. Biol. 2000; 20: 1206-1218Crossref PubMed Scopus (253) Google Scholar). This implies that the N-terminal region of CAF-1 might be specialized as a regulatory domain or in mediating other functions of CAF-1 in DNA damage or heterochromatin maintenance.CAF-1 is recruited to chromatin after DNA damage in human cells, and in vitro, CAF-1 can direct chromatin assembly on many types of damaged DNA templates (16Moggs J.G. Grandi P. Quivy J.P. Jónsson Z.O. Hübscher U. Becker P.B. Almouzni G. Mol. Cell. Biol. 2000; 20: 1206-1218Crossref PubMed Scopus (253) Google Scholar, 17Martini E. Roche D.M. Marheineke K. Verreault A. Almouzni G. J. Cell Biol. 1998; 143: 563-575Crossref PubMed Scopus (144) Google Scholar). Moreover, yeast cells lacking CAF-1 are sensitive to a variety of DNA-damaging agents, including bleomycin, methane methylsulfonate (MMS), and UV radiation (18Kaufman P.D. Kobayashi R. Stillman B. Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (323) Google Scholar, 19Linger J. Tyler J.K. Genetics. 2005; 171: 1513-1522Crossref PubMed Scopus (61) Google Scholar). The BLM helicase also interacts with CAF-1 and inhibits its activity in vitro, and the WRN helicase has been shown to also interact with CAF-1 in a DNA damage-dependent manner (20Jiao R. Bachrati C.Z. Pedrazzi G. Kuster P. Petkovic M. Li J.L. Egli D. Hickson I.D. Stagljar I. Mol. Cell. Biol. 2004; 24: 4710-4719Crossref PubMed Scopus (40) Google Scholar, 21Jiao R. Harrigan J.A. Shevelev I. Dietschy T. Selak N. Indig F.E. Piotrowski J. Janscak P. Bohr V.A. Stagljar I. Oncogene. 2007; 26: 3811-3822Crossref PubMed Scopus (18) Google Scholar). CAF-1 has been shown to be responsible for the deposition of new H3.1 at localized sites of UV or oxidative DNA damage, directly demonstrating that CAF-1 is involved in chromatin assembly at these loci (22Polo S.E. Roche D. Almouzni G. Cell. 2006; 127: 481-493Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). CAF-1 also functions in epigenetic inheritance, most likely through maintenance of histone density in the genome during DNA replication (24Tamburini B.A. Carson J.J. Linger J.G. Tyler J.K. Genetics. 2006; 173: 599-610Crossref PubMed Scopus (19) Google Scholar). 5M. P. Rossmann, W. Luo, O. Tsaponina, A. Chabes, and B. Stillman, manuscript submitted for publication. In yeast, loss of CAF-1 causes defects in the expression of genes near a telomere and at the mating type loci (18Kaufman P.D. Kobayashi R. Stillman B. Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (323) Google Scholar, 25Enomoto S. Berman J. Genes Dev. 1998; 12: 219-232Crossref PubMed Scopus (169) Google Scholar, 26Enomoto S. McCune-Zierath P.D. Gerami-Nejad M. Sanders M.A. Berman J. Genes Dev. 1997; 11: 358-370Crossref PubMed Scopus (137) Google Scholar). These effects are due to aberrant gene expression in cells with low nucleosome density.5 Yeast CAF-1 binds the histone acetyltransferase SAS-1, and human and mouse CAF-1 binds to HP1 proteins and to the methyl-DNA-binding protein MBD1, all of which are involved in the transcriptional silencing of DNA (27Murzina N. Verreault A. Laue E. Stillman B. Mol. Cell. 1999; 4: 529-540Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 28Reese B.E. Bachman K.E. Baylin S.B. Rountree M.R. Mol. Cell. Biol. 2003; 23: 3226-3236Crossref PubMed Scopus (83) Google Scholar). The data from yeast and mammalian systems suggest that one way in which CAF-1 contributes to epigenetic inheritance is by the recruitment of chromatin modifiers to ensure copying of histone marks.We are interested in defining the functions of CAF-1 in vivo and have previously shown that the p150 subunit was essential for cell viability and efficient DNA replication (3Hoek M. Stillman B. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12183-12188Crossref PubMed Scopus (189) Google Scholar). CAF-1 requires other trans factors present in cell extracts to assemble chromatin in addition to PCNA loaded onto the replicated DNA in cis (13Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). We therefore sought to identify factors interacting with CAF-1 under the hypothesis that these proteins may be directly involved in replication-coupled chromatin assembly. CAF-1-interacting factors may also play a role in processes other than DNA replication-coupled chromatin assembly in which CAF-1 functions. In this work, a set of proteins interacting with full-length and a functional, N-terminally truncated CAF-1 p150 were defined by immunoprecipitation-coupled mass spectrometry analysis. Direct interactions between the CAF-1 with the KU complex and with 14-3-3 proteins, both of which are involved in DNA damage responses, were confirmed, suggesting that CAF-1 is centrally involved in histone deposition at multiple types of DNA lesions. This analysis also showed that the previously mapped N-terminal PCNA-interacting region contributes to stable CAF-1-PCNA interaction even though this region is dispensable for chromatin assembly during S phase and DNA damage.DISCUSSIONCAF-1 in yeast has long been linked to the DNA damage response because cells lacking the complex are more sensitive to a variety of DNA-damaging agents. In human cells, CAF-1 is known to interact with the BLM and WRN helicases at sites of prolonged replication fork arrest and is recruited to sites of UV radiation-induced damage to deposit histones de novo (20Jiao R. Bachrati C.Z. Pedrazzi G. Kuster P. Petkovic M. Li J.L. Egli D. Hickson I.D. Stagljar I. Mol. Cell. Biol. 2004; 24: 4710-4719Crossref PubMed Scopus (40) Google Scholar21Jiao R. Harrigan J.A. Shevelev I. Dietschy T. Selak N. Indig F.E. Piotrowski J. Janscak P. Bohr V.A. Stagljar I. Oncogene. 2007; 26: 3811-3822Crossref PubMed Scopus (18) Google Scholar22Polo S.E. Roche D. Almouzni G. Cell. 2006; 127: 481-493Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). We now show that in human cells CAF-1 binds directly to the KU complex that is part of DNA-PK, that CAF-1 is phosphorylated in vitro on its p150 and p60 subunits by this kinase, and that the phosphorylation of CAF-1 by DNA-PK supports an in vitro interaction with 14-3-3 ζ. None of these interactions are important for chromatin assembly as such, and thus the most parsimonious explanation is that they reflect a role of CAF-1 in the DNA damage response.Despite extensive efforts, however, an increased interaction between CAF-1 and either the KU complex or 14-3-3 proteins after DNA damage was not detected in vivo, and no CAF-1-KU complex was seen on DNA in vitro. One possibility is that these interactions are enhanced in the cell after DNA damage but lost after extraction: CAF-1 is tightly bound to chromatin, and only 50% of the protein is salt-extractable and available for study using our approach. Alternatively, the interaction between KU, 14-3-3, and CAF-1 may be related to DNA damage indirectly or to some other cellular process. An indirect relationship between DNA-PK and CAF-1 is supported by a report that the re-expression of CAF-1 in quiescent normal human fibroblasts after bleomycin treatment is dependent upon DNA-PK (4Nabatiyan A. Szüts D. Krude T. Mol. Cell. Biol. 2006; 26: 1839-1849Crossref PubMed Scopus (39) Google Scholar). This study did not determine whether the induction of CAF-1 was at the post-transcriptional level, but it is conceivable that direct phosphorylation of CAF-1 by DNA-PK stabilizes the protein, perhaps in a 14-3-3-dependent manner.14-3-3 proteins have long been linked to the DNA damage response. 14-3-3 σ expression is induced by p53, and it mediates G2 arrest by contributing to the sequestration of CDK1/cyclin B in the cytoplasm (51Chan T.A. Hermeking H. Lengauer C. Kinzler K.W. Vogelstein B. Nature. 1999; 401: 616-620Crossref PubMed Scopus (809) Google Scholar). 14-3-3 proteins in fission yeast bind the checkpoint kinase Chk1 and are important in mediating the DNA damage response (52Peng 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). In budding yeast, 14-3-3 proteins interact with the DNA damage response regulator RAD53 and positively regulate its function, and specific mutants in 14-3-3 proteins have been identified that increase the sensitivity to DNA-damaging agents, perhaps through an effect on global chromatin structure (53Lottersberger F. Panza A. Lucchini G. Longhese M.P. Mol. Cell. Biol. 2007; 27: 3266-3281Crossref PubMed Scopus (20) Google Scholar54Lottersberger F. Rubert F. Baldo V. Lucchini G. Longhese M.P. Genetics. 2003; 165: 1717-1732Crossref PubMed Google Scholar55Usui T. Petrini J.H. Proc. Natl. Acad. Sci. U.S.A. 2007; 104: 2797-2802Crossref PubMed Scopus (33) Google Scholar). Several large scale proteomics analyses have also identified interactions between 14-3-3 proteins and the DNA damage response proteins CHK1, RAD50, and HUS1 in human cells (56Jin J. Smith F.D. Stark C. Wells C.D. Fawcett J.P. Kulkarni S. Metalnikov P. O'Donnell P. Taylor P. Taylor L. Zougman A. Woodgett J.R. Langeberg L.K. Scott J.D. Pawson T. Curr. Biol. 2004; 14: 1436-1450Abstract Full Text Full Text PDF PubMed Scopus (378) Google Scholar, 57Meek S.E. Lane W.S. Piwnica-Worms H. J. Biol. Chem. 2004; 279: 32046-32054Abstract Full Text Full Text PDF PubMed Scopus (185) Google Scholar). It is important to note that the 14-3-3-CAF-1 interaction may have nothing to do with DNA damage, however, and 14-3-3 proteins have many functions in the cell outside of the DNA damage response. The DNA-PK-dependent interaction reflects a requirement for phosphorylation but not necessarily for DNA damage because in vitro treatment with DNA-PK may lead to some nonspecific phosphorylation events.The KU complex is also involved in other cellular functions, including telomere maintenance and potentially the initiation of DNA replication (58d'Adda di Fagagna F. Hande M.P. Tong W.M. Roth D. Lansdorp P.M. Wang Z.Q. Jackson S.P. Curr. Biol. 2001; 11: 1192-1196Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar59Hsu H.L. Gilley D. Galande S.A. Hande M.P. Allen B. Kim S.H. Li G.C. Campisi J. Kohwi-Shigematsu T. Chen D.J. Genes Dev. 2000; 14: 2807-2812Crossref PubMed Scopus (275) Google Scholar60Rampakakis E. Di Paola D. Zannis-Hadjopoulos M. J. Cell Sci. 2008; 121: 590-600Crossref PubMed Scopus (41) Google Scholar). In yeast, the KU complex has recently been shown to be involved in heterochromatin maintenance at the silent mating type loci in part by the recruitment of the silencing regulator Sir4 to epigenetically silenced genes (40Patterson E.E. Fox C.A. Genetics. 2008; 180: 771-783Crossref PubMed Scopus (17) Google Scholar, 61Vandre C.L. Kamakaka R.T. Rivier D.H. Genetics. 2008; 180: 1407-1418Crossref PubMed Scopus (18) Google Scholar). Thus, KU can promote silencing of gene expression.The KU complex is also involved in recovery from a cell cycle checkpoint arrest caused by double-stranded DNA breaks (62Lee S.E. Moore J.K. Holmes A. Umezu K. Kolodner R.D. Haber J.E. Cell. 1998; 94: 399-409Abstract Full Text Full Text PDF PubMed Scopus (645) Google Scholar). Recently, overlapping roles for CAF-1 and ASF1 have been described in turning off the double-stranded DNA break-induced cell cycle checkpoint arrest after repair has occurred (63Kim J.A. Haber J.E. Proc. Natl. Acad. Sci. U.S.A. 2009; 106: 1151-1156Crossref PubMed Scopus (79) Google Scholar). Therefore, the CAF-1-KU interaction may play a role in recruiting CAF-1 to sites of double-stranded DNA break damage to reassemble the appropriate chromatin structure at that site, and once this is achieved, cell cycle progression could occur. Because the global replication defects caused by CAF-1 deficiency globally distort chromatin structure, a definitive answer to this question will require the generation of CAF-1 mutants unable to bind KU.Function of CAF-1 p150 N TerminusCAF-1 p150 has long been reported to have two PCNA binding sites, one at the N terminus (PIP1), which is very robust when attached to GST for in vitro binding assays, and one that is internal (PIP2; Refs. 5Ye X. Franco A.A. Santos H. Nelson D.M. Kaufman P.D. Adams P.D. Mol. Cell. 2003; 11: 341-351Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar, 15Krawitz D.C. Kama T. Kaufman P.D. Mol. Cell. Biol. 2002; 22: 614-625Crossref PubMed Scopus (135) Google Scholar, and 16Moggs J.G. Grandi P. Quivy J.P. Jónsson Z.O. Hübscher U. Becker P.B. Almouzni G. Mol. Cell. Biol. 2000; 20: 1206-1218Crossref PubMed Scopus (253) Google Scholar). The PIP2 PCNA binding region is essential because it is conserved from yeast to human and is necessary to CAF-1 function in vivo for recruiting CAF-1 to sites of DNA replication and in vitro chromatin assembly assays (12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar). The role of the PIP1 binding site is not known. We now conclusively show that the N-terminal PCNA binding site is involved in PCNA binding in the cell but is dispensable for normal CAF-1 functions in DNA replication- and damage-coupled assembly. Similar results with N-terminally truncated CAF-1 p150 have also been reported using a conditional knock-out approach in chicken DT40 cells, although PCNA binding to CAF-1 was reportedly unaffected in this cell line (64Takami Y. Ono T. Fukagawa T. Shibahara K. Nakayama T. Mol. Biol. Cell. 2007; 18: 129-141Crossref PubMed Scopus (68) Google Scholar).Other CAF-1-interacting ProteinsAlthough we identified numerous novel CAF-1-interacting proteins in this analysis, we did not identify some previously identified interactors such as ASF1, MDB1, BLM, and WRN. MBD1 and BLM were identified as CAF-1-interacting proteins on the basis of two-hybrid screens and may thus interact with only a small minority of CAF-1 in the cell and be below the threshold of detection. Direct interactions between human ASF1 and CAF-1 have been demonstrated, and ASF1 is a component of the soluble H3.1 and H3.3 complexes (23Mello J.A. Silljé H.H. Roche D.M. Kirschner D.B. Nigg E.A. Almouzni G. EMBO Rep. 2002; 3: 329-334Crossref PubMed Scopus (227) Google Scholar, 43Tagami H. Ray-Gallet D. Almouzni G. Nakatani Y. Cell. 2004; 116: 51-61Abstract Full Text Full Text PDF PubMed Scopus (968) Google Scholar). It may be that the ASF1-H3/H4 complex is largely separate from the CAF-1-H3/H4 complex in the cell and functions to deliver histones to CAF-1. Indeed, there are several lines of biochemical evidence that suggest that this may be one function of ASF1 in human cells (50Groth A. Ray-Gallet D. Quivy J.P. Lukas J. Bartek J. Almouzni G. Mol. Cell. 2005; 17: 301-311Abstract Full Text Full Text PDF PubMed Scopus (197) Google Scholar). If this were true, only a small amount of CAF-1 might bind ASF1 at any particular time, perhaps below the detection limit of our affinity purification approach that involved extraction of CAF-1 off chromatin with high salt. Seen in this light, the proteins that were identified in this screen might represent factors that are bound to a significant percentage of CAF-1 albeit dynamically. It will thus be interesting to further investigate their function in the context of CAF-1 by identifying mutant versions of CAF-1 that have specific defects in binding one or more of these proteins. IntroductionDNA in eukaryotic cells is packaged into nucleosomes that form higher order chromatin. During DNA replication, histones H3 and H4 that form the core of the nucleosome are randomly partitioned to the daughter strands, and new histones are deposited de novo to maintain histone density in the wake of the replication fork (1Verreault A. Genes Dev. 2000; 14: 1430-1438PubMed Google Scholar). Although many histone chaperones exist in human cells, the primary DNA replication-coupled histone deposition factor is CAF-1 as determined from biochemical purification experiments (2Smith S. Stillman B. Cell. 1989; 58: 15-25Abstract Full Text PDF PubMed Scopus (513) Google Scholar). Depletion of CAF-1 from human cells has been shown to lead to defects in DNA replication fork progression, activation of the intra-S phase checkpoint, and global defects in chromatin structure (3Hoek M. Stillman B. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12183-12188Crossref PubMed Scopus (189) Google Scholar4Nabatiyan A. Szüts D. Krude T. Mol. Cell. Biol. 2006; 26: 1839-1849Crossref PubMed Scopus (39) Google Scholar5Ye X. Franco A.A. Santos H. Nelson D.M. Kaufman P.D. Adams P.D. Mol. Cell. 2003; 11: 341-351Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar).CAF-1 is a heterotrimeric complex consisting of 150-, 60-, and 48-kDa subunits that binds to histones H3 and H4 (6Kaufman P.D. Kobayashi R. Kessler N. Stillman B. Cell. 1995; 81: 1105-1114Abstract Full Text PDF PubMed Scopus (307) Google Scholar, 7Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Cell. 1996; 87: 95-104Abstract Full Text Full Text PDF PubMed Scopus (521) Google Scholar). The p60 and p48 subunits consist largely of WD40 repeats that play a role in histone binding, and p48 is known to be a member of numerous H3/H4 binding complexes such as HAT1 and HDAC1 (8Ahmad A. Takami Y. Nakayama T. J. Biol. Chem. 1999; 274: 16646-16653Abstract Full Text Full Text PDF PubMed Scopus (31) Google Scholar9Tie F. Furuyama T. Prasad-Sinha J. Jane E. Harte P.J. Development. 2001; 128: 275-286Crossref PubMed Google Scholar10Verreault A. Kaufman P.D. Kobayashi R. Stillman B. Curr. Biol. 1998; 8: 96-108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar). CAF-1 p60 also interacts with the histone chaperones ASF1A and ASF1B through their B-domain, which resides outside of the WD40 repeat region (11Tang Y. Poustovoitov M.V. Zhao K. Garfinkel M. Canutescu A. Dunbrack R. Adams P.D. Marmorstein R. Nat. Struct. Mol. Biol. 2006; 13: 921-929Crossref PubMed Scopus (131) Google Scholar). The p150 subunit has been better characterized and contains the regions that mediate interactions with proliferating cell nuclear antigen (PCNA), 4The abbreviations used are: PCNA, proliferating cell nuclear antigen; DNA-PK, DNA-dependent protein kinase; MMS, methane methylsulfonate; DNA-PKCS, DNA-dependent protein kinase catalytic subunit; LC-MS/MS, liquid chromatography-tandem mass spectrometry; CDK, cyclin-dependent kinase. HP1, MBD1, BLM, and the CAF-1 p60 subunit. The PCNA mediates the connection between CAF-1 and the DNA replication fork (12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar13Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar14Zhang Z. Shibahara K. Stillman B. Nature. 2000; 408: 221-225Crossref PubMed Scopus (384) Google Scholar). There are two PCNA binding sites that have been mapped in the p150 subunit, but CAF-1 is localized to replication forks through a conserved PCNA binding region motif called PIP2 in the middle of the p150 open reading frame (5Ye X. Franco A.A. Santos H. Nelson D.M. Kaufman P.D. Adams P.D. Mol. Cell. 2003; 11: 341-351Abstract Full Text Full Text PDF PubMed Scopus (204) Google Scholar, 12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar, 15Krawitz D.C. Kama T. Kaufman P.D. Mol. Cell. Biol. 2002; 22: 614-625Crossref PubMed Scopus (135) Google Scholar). The N terminus of p150, containing a second PCNA binding region as well as the HP1-interacting region, is dispensable for DNA replication-coupled CAF-1 chromatin assembly activity in vitro and for localization to replication foci in vivo (12Rolef Ben-Shahar T. Castillo A.G. Osborne M.J. Borden K.L. Kornblatt J. Verreault A. Mol. Cell. Biol. 2009; 29: 6353-6365Crossref PubMed Scopus (47) Google Scholar, 16Moggs J.G. Grandi P. Quivy J.P. Jónsson Z.O. Hübscher U. Becker P.B. Almouzni G. Mol. Cell. Biol. 2000; 20: 1206-1218Crossref PubMed Scopus (253) Google Scholar). This implies that the N-terminal region of CAF-1 might be specialized as a regulatory domain or in mediating other functions of CAF-1 in DNA damage or heterochromatin maintenance.CAF-1 is recruited to chromatin after DNA damage in human cells, and in vitro, CAF-1 can direct chromatin assembly on many types of damaged DNA templates (16Moggs J.G. Grandi P. Quivy J.P. Jónsson Z.O. Hübscher U. Becker P.B. Almouzni G. Mol. Cell. Biol. 2000; 20: 1206-1218Crossref PubMed Scopus (253) Google Scholar, 17Martini E. Roche D.M. Marheineke K. Verreault A. Almouzni G. J. Cell Biol. 1998; 143: 563-575Crossref PubMed Scopus (144) Google Scholar). Moreover, yeast cells lacking CAF-1 are sensitive to a variety of DNA-damaging agents, including bleomycin, methane methylsulfonate (MMS), and UV radiation (18Kaufman P.D. Kobayashi R. Stillman B. Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (323) Google Scholar, 19Linger J. Tyler J.K. Genetics. 2005; 171: 1513-1522Crossref PubMed Scopus (61) Google Scholar). The BLM helicase also interacts with CAF-1 and inhibits its activity in vitro, and the WRN helicase has been shown to also interact with CAF-1 in a DNA damage-dependent manner (20Jiao R. Bachrati C.Z. Pedrazzi G. Kuster P. Petkovic M. Li J.L. Egli D. Hickson I.D. Stagljar I. Mol. Cell. Biol. 2004; 24: 4710-4719Crossref PubMed Scopus (40) Google Scholar, 21Jiao R. Harrigan J.A. Shevelev I. Dietschy T. Selak N. Indig F.E. Piotrowski J. Janscak P. Bohr V.A. Stagljar I. Oncogene. 2007; 26: 3811-3822Crossref PubMed Scopus (18) Google Scholar). CAF-1 has been shown to be responsible for the deposition of new H3.1 at localized sites of UV or oxidative DNA damage, directly demonstrating that CAF-1 is involved in chromatin assembly at these loci (22Polo S.E. Roche D. Almouzni G. Cell. 2006; 127: 481-493Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). CAF-1 also functions in epigenetic inheritance, most likely through maintenance of histone density in the genome during DNA replication (24Tamburini B.A. Carson J.J. Linger J.G. Tyler J.K. Genetics. 2006; 173: 599-610Crossref PubMed Scopus (19) Google Scholar). 5M. P. Rossmann, W. Luo, O. Tsaponina, A. Chabes, and B. Stillman, manuscript submitted for publication. In yeast, loss of CAF-1 causes defects in the expression of genes near a telomere and at the mating type loci (18Kaufman P.D. Kobayashi R. Stillman B. Genes Dev. 1997; 11: 345-357Crossref PubMed Scopus (323) Google Scholar, 25Enomoto S. Berman J. Genes Dev. 1998; 12: 219-232Crossref PubMed Scopus (169) Google Scholar, 26Enomoto S. McCune-Zierath P.D. Gerami-Nejad M. Sanders M.A. Berman J. Genes Dev. 1997; 11: 358-370Crossref PubMed Scopus (137) Google Scholar). These effects are due to aberrant gene expression in cells with low nucleosome density.5 Yeast CAF-1 binds the histone acetyltransferase SAS-1, and human and mouse CAF-1 binds to HP1 proteins and to the methyl-DNA-binding protein MBD1, all of which are involved in the transcriptional silencing of DNA (27Murzina N. Verreault A. Laue E. Stillman B. Mol. Cell. 1999; 4: 529-540Abstract Full Text Full Text PDF PubMed Scopus (256) Google Scholar, 28Reese B.E. Bachman K.E. Baylin S.B. Rountree M.R. Mol. Cell. Biol. 2003; 23: 3226-3236Crossref PubMed Scopus (83) Google Scholar). The data from yeast and mammalian systems suggest that one way in which CAF-1 contributes to epigenetic inheritance is by the recruitment of chromatin modifiers to ensure copying of histone marks.We are interested in defining the functions of CAF-1 in vivo and have previously shown that the p150 subunit was essential for cell viability and efficient DNA replication (3Hoek M. Stillman B. Proc. Natl. Acad. Sci. U.S.A. 2003; 100: 12183-12188Crossref PubMed Scopus (189) Google Scholar). CAF-1 requires other trans factors present in cell extracts to assemble chromatin in addition to PCNA loaded onto the replicated DNA in cis (13Shibahara K. Stillman B. Cell. 1999; 96: 575-585Abstract Full Text Full Text PDF PubMed Scopus (535) Google Scholar). We therefore sought to identify factors interacting with CAF-1 under the hypothesis that these proteins may be directly involved in replication-coupled chromatin assembly. CAF-1-interacting factors may also play a role in processes other than DNA replication-coupled chromatin assembly in which CAF-1 functions. In this work, a set of proteins interacting with full-length and a functional, N-terminally truncated CAF-1 p150 were defined by immunoprecipitation-coupled mass spectrometry analysis. Direct interactions between the CAF-1 with the KU complex and with 14-3-3 proteins, both of which are involved in DNA damage responses, were confirmed, suggesting that CAF-1 is centrally involved in histone deposition at multiple types of DNA lesions. This analysis also showed that the previously mapped N-terminal PCNA-interacting region contributes to stable CAF-1-PCNA interaction even though this region is dispensable for chromatin assembly during S phase and DNA damage.

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