MCB Accepts, published online ahead of print on 12 October 2009

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Mol. Cell. Biol. doi:10.1128/MCB.01051-09
Copyright (c) 2009, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Two Fundamentally Distinct PCNA-Interaction Peptides Contribute to Chromatin Assembly Factor 1 Function

Tom Ben-Shahar, Araceli G. Castillo, Michael J. Osborne, Katherine L. B. Borden, Jack Kornblatt, and Alain Verreault^*

Cancer Research UK, London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, United Kingdom; Departamento de Genética, Facultad de Biologia, Universitad de Malaga, 29010 Malaga, Spain; Department of Biology, Concordia University, Montréal (Québec), H4B 1R6, Canada; Institute for Research in Immunology and Cancer (IRIC), Département de Pathologie et Biologie Cellulaire, Université de Montréal, CP 6128, Succursale Centre-Ville, Montréal (Québec), H3C 3J7, Canada

^* To whom correspondence should be addressed. Email: alain.verreault{at}umontreal.ca.

Chromatin Assembly Factor 1 (CAF-1) deposits histones H3 and H4 rapidly behind replication forks through an interaction with the Proliferating Cell Nuclear Antigen (PCNA), a DNA polymerase processivity factor that also binds to a number of replication enzymes and other proteins that act on nascent DNA. The mechanisms that enable CAF-1 and other PCNA-binding proteins to function harmoniously at the replication fork are poorly understood. Here we report that the large subunit of human CAF-1 (p150) contains two distinct PCNA-interaction peptides (PIPs). The N-terminal PIP binds strongly to PCNA in vitro but, surprisingly, is dispensable for nucleosome assembly and only makes a modest contribution to targeting p150 to DNA replication foci in vivo. In contrast, the internal PIP (PIP2) lacks one of the highly conserved residues of canonical PIPs and binds weakly to PCNA. Surprisingly, PIP2 is essential for nucleosome assembly during DNA replication in vitro and plays a major role in targeting p150 to sites of DNA replication. Unlike canonical PIPs, such as that of p21, the two p150 PIPs are capable of preferentially inhibiting nucleosome assembly, rather than DNA synthesis, suggesting that intrinsic features of these peptides are part of the mechanism that enables CAF-1 to function behind replication forks without interfering with other PCNA-mediated processes.