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MBC in Press, published online ahead of print October 25, 2006
Mol. Biol. Cell 10.1091/mbc.E06-05-0426

A more recent version of this article appeared on January 1, 2007
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Submitted on May 18, 2006
Revised on October 10, 2006
Accepted on October 13, 2006

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

Yasunari Takami,* Tatsuya Ono,{dagger} Tatsuo Fukagawa,{ddagger} Kei-ichi Shibahara,{dagger} and Tatsuo Nakayama*{sect}

*Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, and {sect}Department of Life Science, Frontier Science Research Center, University of Miyazaki, 5200 Kihara, Kiyotake, Miyazaki 889-1692, Japan; {dagger}Departments of Integrated Genetics and {ddagger}Molecular Genetics, National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan

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 human, 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, PCNA, but not with heterochromatin protein, HP1-{gamma}, 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.

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




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