Lee S H, Kim D K
Department of Virology and Molecular Biology, St. Jude Children's Research Hospital, Memphis, Tennessee 38101-0318, USA.
J Biol Chem. 1995 May 26;270(21):12801-7. doi: 10.1074/jbc.270.21.12801.
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.
人类复制蛋白A（RPA）是一种由三个亚基组成的蛋白复合物，参与DNA复制、修复和重组过程。我们通过构建一系列p34突变体，研究了RPA的34-kDa亚基（p34）在DNA复制中的作用。虽然缺失p34的N端结构域可阻止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被DNA依赖性激酶磷酸化。在猴病毒40（SV40）DNA复制中，含有上述任何一种突变体的RPA的功能与野生型RPA一样高效。然而，含有C端缺失p34的突变体RPA仅有微弱活性。这表明，对于RPA在DNA复制中的功能而言，p34的C端区域而非磷酸化结构域是必需的。此外，含有C端缺失p34突变体的RPA可刺激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）相互作用的能力有限。这些结果表明，在SV40 DNA复制起始过程中，RPA p34与SV40 T Ag相互作用，可能对DNA解旋是必需的。
