Henricksen L A, Wold M S
Department of Biochemistry, University of Iowa College of Medicine, Iowa City 52242.
J Biol Chem. 1994 Sep 30;269(39):24203-8.
Replication Protein A (RPA) is a multisubunit, single-stranded DNA-binding protein essential for DNA metabolism in eukaryotic cells. The 32-kDa subunit of RPA is phosphorylated in a cell cycle-dependent manner becoming phosphorylated during S phase. It has been postulated that this phosphorylation may regulate the activities of RPA and that the family of p34cdc2 kinases directly catalyzes the phosphorylation of RPA in the cell. We have mutated the two consensus p34cdc2 sites in the 32-kDa subunit of RPA individually and in combination and purified the mutant protein complexes. Mutant RPA with both consensus p34cdc2 sites converted to alanine was not phosphorylated by purified p34cdc2 kinase. Nevertheless, we found that the properties of these RPA mutants were identical to those of the wild-type protein. The mutated RPA proteins had normal single-stranded DNA binding activity and were completely functional for DNA replication. In addition, the mutants became hyperphosphorylated when incubated under DNA replication conditions. These results demonstrate that phosphorylation by p34cdc2 kinase is not essential for RPA function in DNA replication in vitro. Possible roles of RPA phosphorylation on DNA metabolism are discussed.
复制蛋白A（RPA）是一种多亚基单链DNA结合蛋白，对真核细胞中的DNA代谢至关重要。RPA的32 kDa亚基在细胞周期依赖性方式下发生磷酸化，在S期被磷酸化。据推测，这种磷酸化可能调节RPA的活性，并且p34cdc2激酶家族直接催化细胞中RPA的磷酸化。我们分别和联合突变了RPA 32 kDa亚基中的两个共有p34cdc2位点，并纯化了突变蛋白复合物。两个共有p34cdc2位点都转化为丙氨酸的突变RPA不会被纯化的p34cdc2激酶磷酸化。然而，我们发现这些RPA突变体的特性与野生型蛋白相同。突变的RPA蛋白具有正常的单链DNA结合活性，并且对DNA复制完全有功能。此外，当在DNA复制条件下孵育时，突变体发生超磷酸化。这些结果表明，p34cdc2激酶的磷酸化对于体外DNA复制中RPA的功能不是必需的。讨论了RPA磷酸化对DNA代谢的可能作用。
