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首页> 外文期刊>Molecular and Cellular Biology >Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers
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Replication Protein A (RPA) Phosphorylation Prevents RPA Association with Replication Centers

机译：复制蛋白A（RPA）磷酸化可防止RPA与复制中心关联

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Mammalian replication protein A (RPA) undergoes DNA damage-dependent phosphorylation at numerous sites on the N terminus of the RPA2 subunit. To understand the functional significance of RPA phosphorylation, we expressed RPA2 variants in which the phosphorylation sites were converted to aspartate (RPA2D) or alanine (RPA2A). Although RPA2D was incorporated into RPA heterotrimers and supported simian virus 40 DNA replication in vitro, the RPA2D mutant was selectively unable to associate with replication centers in vivo. In cells containing greatly reduced levels of endogenous RPA2, RPA2D again did not localize to replication sites, indicating that the defect in supporting chromosomal DNA replication is not due to competition with the wild-type protein. Use of phosphospecific antibodies demonstrated that endogenous hyperphosphorylated RPA behaves similarly to RPA2D. In contrast, under DNA damage or replication stress conditions, RPA2D, like RPA2A and wild-type RPA2, was competent to associate with DNA damage foci as determined by colocalization with γ-H2AX. We conclude that RPA2 phosphorylation prevents RPA association with replication centers in vivo and potentially serves as a marker for sites of DNA damage.
机译：哺乳动物复制蛋白A（RPA）在RPA2亚基N末端的许多位点经历DNA损伤依赖性磷酸化。为了了解RPA磷酸化的功能意义，我们表达了RPA2变异体，其中的磷酸化位点被转化为天冬氨酸（RPA2 D ）或丙氨酸（RPA2 A ）。尽管将RPA2 D 掺入RPA异源三聚体并支持猿猴病毒40 DNA的体外复制，但RPA2 D 突变体选择性地无法与体内复制中心结合。在内源性RPA2水平大大降低的细胞中，RPA2 D 再次未定位于复制位点，表明支持染色体DNA复制的缺陷不是由于与野生型蛋白竞争。磷酸化特异性抗体的使用表明内源性超磷酸化RPA的行为类似于RPA2 D 。相反，在DNA损伤或复制胁迫条件下，RPA2 D （如RPA2 A ）和野生型RPA2能够与DNA损伤灶相关联（通过与γ-H2AX。我们得出的结论是，RPA2磷酸化可防止RPA与体内复制中心缔合，并有可能充当DNA损伤位点的标记。

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