Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein A complex formation
Novel checkpoint response to genotoxic stress mediated by nucleolin-replication protein A complex formation
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DOI：
10.1128/mcb.25.6.2463-2474.2005
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发表时间：
2005-03-01
影响因子：
5.3
通讯作者：
Borowiec, JA
中科院分区：
文献类型：
--
作者：
Kim, K;Dimitrova, DD;Borowiec, JA
人类复制蛋白A （RPA）是一种主要的单链dna结合蛋白，在热休克后被发现与核蛋白形成复合物而受到抑制。在这里，我们表明核素- rpa复合物的形成在基因毒性应激（如喜树碱处理或暴露于电离辐射）后受到刺激。体外和体内复合物的形成需要一个位于核蛋白C末端的63个残基的富含甘氨酸精氨酸（GAR）结构域，该结构域足以在体外抑制DNA复制。荧光共振能量转移研究表明，胁迫后核蛋白- ppa相互作用既发生在核质中，也发生在核仁中。GAR结构域或与RPA构成相互作用的核仁蛋白突变体（TM）的表达足以抑制进入S期。通过过表达RPA2亚基来增加细胞RPA水平可以使核蛋白GAR或TM表达对染色体DNA复制的抑制作用最小化。这种阻滞与ATM或ATR对p53的激活无关，也不涉及p21的表达升高。我们的数据揭示了一种新的细胞机制，通过抑制一种必需的DNA复制因子来抑制基因组复制以响应基因毒性应激。
Human replication protein A (RPA), the primary single-stranded DNA-binding protein, was previously found to be inhibited after heat shock by complex formation with nucleolin. Here we show that nucleolin-RPA complex formation is stimulated after genotoxic stresses such as treatment with camptothecin or exposure to ionizing radiation. Complex formation in vitro and in vivo requires a 63-residue glycine-arginine-rich (GAR) domain located at the extreme C terminus of nucleolin, with this domain sufficient to inhibit DNA replication in vitro. Fluorescence resonance energy transfer studies demonstrate that the nucleolin-PPA interaction after stress occurs both in the nucleoplasm and in the nucleolus. Expression of the GAR domain or a nucleolin mutant (TM) with a constitutive interaction with RPA is sufficient to inhibit entry into S phase. Increasing cellular RPA levels by overexpression of the RPA2 subunit minimizes the inhibitory effects of nucleolin GAR or TM expression on chromosomal DNA replication. The arrest is independent of p53 activation by ATM or ATR and does not involve heightened expression of p21. Our data reveal a novel cellular mechanism that represses genomic replication in response to genotoxic stress by inhibition of an essential DNA replication factor.