Essential role of chromatin assembly factor-1-mediated rapid nucleosome assembly for DNA replication and cell division in vertebrate cells
Essential role of chromatin assembly factor-1-mediated rapid nucleosome assembly for DNA replication and cell division in vertebrate cells
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DOI：
10.1091/mbc.e06-05-0426
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发表时间：
2007-01-01
影响因子：
3.3
通讯作者：
Nakayama, Tatsuo
中科院分区：
文献类型：
--
作者：
Takami, Yasunari;Ono, Tatsuya;Nakayama, Tatsuo
染色质组装因子-1（CAF-1）是由p150、p60和p48亚基组成的复合物，从酵母到人类高度保守，并促进体外新复制DNA的核小体组装。为了研究CAF-1在脊椎动物中的作用，我们产生了两个条件性DT 40突变体，分别缺乏CAF-1 p150和p60。这些CAF-1亚基中的每一个的消耗导致S期进展延迟，伴随着缓慢的DNA合成，随后在S/G（2）晚期积累，与额外的中心体相关的异常有丝分裂，然后最终的结果是细胞死亡。我们证明，CAF-1是必要的快速核小体形成过程中DNA复制在体内以及在体外。CAF-1的缺失与S-检查点激酶Chk 1和Chk 2磷酸化的明显诱导无关。为了阐明CAF-1 p150中结构域的确切作用，进行了包括拯救测定的功能解剖分析。结果表明，CAF-1 p150与CAF-1 p60和DNA聚合酶滑动钳增殖细胞核抗原（PCNA）的结合能力，而不是与异染色质蛋白HP 1-γ的结合能力是细胞存活所必需的。这些观察结果强调了CAF-1依赖的核小体组装通过其与PCNA的相互作用在DNA复制和细胞增殖中的重要作用。
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/G(2), 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.