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Vol. 18, Issue 1, 129-141, January 2007

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Essential Role of Chromatin Assembly Factor-1–mediated Rapid Nucleosome Assembly for DNA Replication and Cell Division in Vertebrate Cells

Yasunari Takami^*, Tatsuya Ono, Tatsuo Fukagawa, Kei-ichi Shibahara, and Tatsuo Nakayama^*,

*Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan; Department of Life Science, Frontier Science Research Center, University of Miyazaki, Miyazaki 889-1692, Japan; and Departments of Integrated Genetics and Molecular Genetics, National Institute of Genetics, Shizuoka 411-8540, Japan

Submitted May 18, 2006; Revised October 10, 2006; Accepted October 13, 2006
Monitoring Editor: A. Gregory Matera

Chromatin assembly factor-1 (CAF-1), a complex consisting of p150, p60, and p48 subunits, is highly conserved from yeast to humans and facilitates nucleosome assembly of newly replicated DNA in vitro. To investigate roles of CAF-1 in vertebrates, we generated two conditional DT40 mutants, respectively, devoid of CAF-1p150 and p60. Depletion of each of these CAF-1 subunits led to delayed S-phase progression concomitant with slow DNA synthesis, followed by accumulation in late S/G₂ phase and aberrant mitosis associated with extra centrosomes, and then the final consequence was cell death. We demonstrated that CAF-1 is necessary for rapid nucleosome formation during DNA replication in vivo as well as in vitro. Loss of CAF-1 was not associated with the apparent induction of phosphorylations of S-checkpoint kinases Chk1 and Chk2. To elucidate the precise role of domain(s) in CAF-1p150, functional dissection analyses including rescue assays were preformed. Results showed that the binding abilities of CAF-1p150 with CAF-1p60 and DNA polymerase sliding clamp proliferating cell nuclear antigen (PCNA) but not with heterochromatin protein HP1- are required for cell viability. These observations highlighted the essential role of CAF-1–dependent nucleosome assembly in DNA replication and cell proliferation through its interaction with PCNA.

This article was published online ahead of print in MBC in Press (http://www.molbiolcell.org/cgi/doi/10.1091/mbc.E06-05-0426) on October 25, 2006.

Address correspondence to: Tatsuo Nakayama (tnakayam{at}med.miyazaki-u.ac.jp)

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