An analysis of CAF-1 interacting proteins reveals dynamic and direct interactions with the ku complex and 14-3-3 proteins

Hoek, M., Myers, M. P., Stillman, B. (2011) An analysis of CAF-1 interacting proteins reveals dynamic and direct interactions with the ku complex and 14-3-3 proteins. Journal of Biological Chemistry, 286 (12). pp. 10876-10887. ISSN 0021-9258

URL: http://www.ncbi.nlm.nih.gov/pubmed/21209461
DOI: 10.1074/jbc.M110.217075

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 genes expression and in chromatin assembly following DNA repair. In this study, we have generated cell lines expressing RNAi resistant versions of CAF-1 and show that the N-terminal 296 amino acids are dispensable for essential CAF-1 function in vivo. N-terminally truncated CAF-1 p150 is deficient in PCNA binding, reinforcing the existence of two PCNA binding sites in human CAF-1, but the defect in PCNA binding has 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 reveals that CAF-1, is directly associated with the Ku70/80 complex, part of the DNA dependent protein kinase, and the phospho-serine/threonine binding protein, 14-3-3 zeta. CAF-1 is a substrate for DNA-PK and the 14-3-3 interaction in vitro is dependent on DNA-PK 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.

Item Type: Paper
Uncontrolled Keywords: Proteins/genetics Proteins/metabolism Antigens, Nuclear/genetics Antigens, Nuclear/metabolism Cell Line Chromatin Assembly Factor-1/genetics Chromatin Assembly Factor-1/metabolism Chromatin Assembly and Disassembly/physiology DNA Damage/physiology DNA Repair/physiology DNA Replication/physiology DNA-Binding Proteins/genetics DNA-Binding Proteins/metabolism Histones/genetics Histones/metabolism Humans Multiprotein Complexes/genetics Multiprotein Complexes/metabolism Proliferating Cell Nuclear Antigen/genetics Proliferating Cell Nuclear Antigen/metabolism Protein Binding
Subjects: bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > DNA replication
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > RNAi
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > Chromatin dynamics
bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > histone
CSHL Authors:
Communities: CSHL labs > Stillman lab
CSHL Cancer Center Program > Gene Regulation and Cell Proliferation
Highlight: Stillman, Bruce W.
Depositing User: CSHL Librarian
Date Deposited: 09 Nov 2011 19:34
Last Modified: 20 Jun 2017 16:31
PMCID: PMC3060538
Related URLs:
URI: http://repository.cshl.edu/id/eprint/15646

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