title: An analysis of CAF-1-interacting proteins reveals dynamic and direct interactions with the KU complex and 14-3-3 proteins.
abstract: 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.
authors.affiliation: Cold Spring Harbor Laboratory, Cold Spring Harbor, New York 11724, USA.
authors.forename: Maarten
authors.initials: M
authors.lastname: Hoek
authors.forename: Michael P
authors.initials: MP
authors.lastname: Myers
authors.forename: Bruce
authors.initials: B
authors.lastname: Stillman
mesh_terms: D048948:14-3-3 Proteins / Q000235:genetics / Q000378:metabolism*; D034961:Antigens, Nuclear / Q000235:genetics / Q000378:metabolism*; D002460:Cell Line; D056504:Chromatin Assembly Factor-1 / Q000235:genetics / Q000378:metabolism*; D042002:Chromatin Assembly and Disassembly / Q000502:physiology*; D004249:DNA Damage / Q000502:physiology; D004260:DNA Repair / Q000502:physiology*; D004261:DNA Replication / Q000502:physiology*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D000072200:Ku Autoantigen; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding
chemical_list: D048948:14-3-3 Proteins; D034961:Antigens, Nuclear; D056504:Chromatin Assembly Factor-1; D004268:DNA-Binding Proteins; D006657:Histones; D046912:Multiprotein Complexes; D018809:Proliferating Cell Nuclear Antigen; C482984:YWHAH protein, human; C000606509:Xrcc6 protein, human; D000072200:Ku Autoantigen