THE ROLE OF THE 34-KDA SUBUNIT OF HUMAN REPLICATION PROTEIN-A IN SIMIAN-VIRUS-40 DNA-REPLICATION IN-VITRO
THE ROLE OF THE 34-KDA SUBUNIT OF HUMAN REPLICATION PROTEIN-A IN SIMIAN-VIRUS-40 DNA-REPLICATION IN-VITRO
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DOI：
10.1074/jbc.270.21.12801
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发表时间：
1995-05-26
影响因子：
4.8
通讯作者：
KIM, DK
中科院分区：
文献类型：
--
作者：
LEE, SH;KIM, DK
人类复制蛋白 A (RPA) 是一种三亚基蛋白复合物，参与 DNA 复制、修复和重组。我们通过生成一系列 p34 突变体研究了 RPA 34 kDa 亚基 (p34) 在 DNA 复制中的作用。虽然 p34 N 端结构域的缺失阻止了其被细胞周期蛋白依赖性激酶 (Cdk) 和 DNA 依赖性激酶磷酸化，但缺乏 Cdk 主要磷酸化位点的双点突变体可能会被 DNA 依赖性激酶磷酸化。在猿猴病毒 40 (SV40) DNA 复制中，含有这些突变体的 RPA 与野生型 RPA 一样有效地发挥作用。然而，含有 C 末端删除的 p34 的突变 RPA 仅具有轻微活性。这表明 C 末端区域（而非 p34 的磷酸化结构域）对于 DNA 复制中的 RPA 功能是必需的。此外，含有C端缺失的p34突变体的RPA可以刺激DNA聚合酶α，并与单链DNA结合，但其解旋DNA或与SV40大T抗原（T Ag）相互作用的能力受到限制。这些结果表明，RPA p34 在 SV40 DNA 复制起始过程中与 SV40 T Ag 相互作用，并且可能是 DNA 解旋所必需的。
Human replication protein A (RPA) is a three subunit protein complex involved in DNA replication, repair, and recombination. We investigated the role of the 34-kDa subunit (p34) of RPA in DNA replication by generating a series of p34 mutants. While deletion of the N-terminal domain of p34 prevented its phosphorylation by both cyclin dependent kinase (Cdk) and DNA-dependent kinase, a double point mutant that lacks the major phosphorylation sites for Cdk could be phosphorylated by DNA-dependent kinase. In simian virus 40 (SV40) DNA replication, RPA containing either of these mutants functioned as efficiently as wild-type RPA. However, mutant RPA containing C-terminally deleted p34 was only marginally active. This indicates that the C-terminal region, but not the phosphorylation domain of p34, is necessary for RPA function in DNA replication. Furthermore, RPA containing the C-terminally deleted p34 mutant could stimulate DNA polymerase alpha, and bind to single-stranded DNAs but was limited in its ability to unwind DNA or interact with SV40 large T antigen (T Ag). These results suggest that RPA p34 interacts with SV40 T Ag during the initiation of SV40 DNA replication and may be necessary for DNA unwinding.