Essential role of chromatin assembly factor-1-mediated rapid nucleosome assembly for DNA replication and cell division in vertebrate cells.
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-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.
Takami
Yasunari
Y
Section of Biochemistry and Molecular Biology, Department of Medical Sciences, Miyazaki Medical College, University of Miyazaki, Miyazaki 889-1692, Japan.
Ono
Tatsuya
T
Fukagawa
Tatsuo
T
Shibahara
Kei-ichi
K
Nakayama
Tatsuo
T
D000818:Animals; D002470:Cell Survival; D018385:Centrosome / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D002645:Chickens; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone / Q000172:deficiency / Q000378:metabolism*; D004261:DNA Replication; D004268:DNA-Binding Proteins / Q000172:deficiency / Q000378:metabolism*; D004789:Enzyme Activation; D018390:Gene Targeting; D006801:Humans; D008938:Mitosis; D009707:Nucleosomes / Q000378:metabolism*; D018809:Proliferating Cell Nuclear Antigen / Q000378:metabolism; D011485:Protein Binding; D011494:Protein Kinases / Q000378:metabolism; D021122:Protein Subunits / Q000378:metabolism; D016196:S Phase; D008941:Spindle Apparatus / Q000378:metabolism; D014157:Transcription Factors; D014714:Vertebrates / Q000378:metabolism*
C533593:CHAF1B protein, human; C489780:CNOT8 protein, human; D056504:Chromatin Assembly Factor-1; D002868:Chromosomal Proteins, Non-Histone; D004268:DNA-Binding Proteins; D009707:Nucleosomes; D018809:Proliferating Cell Nuclear Antigen; D021122:Protein Subunits; D014157:Transcription Factors; D011494:Protein Kinases; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1