REPLICATION PROTEIN-A MUTANTS LACKING PHOSPHORYLATION SITES FOR P34(CDC2) KINASE SUPPORT DNA-REPLICATION

Replication Protein A (RPA) is a multisubunit, single-stranded DNA-bin ding 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 th is phosphorylation may regulate the activities of RPA and that the fam ily of p34(cdc2) kinases directly catalyzes the phosphorylation of RPA in the cell, We have mutated the two consensus p34(cdc2) sites in the 32-kDa subunit of RPA individually and in combination and purified th e mutant protein complexes. Mutant RPA with both consensus p34(cdc2) s ites converted to alanine was not phosphorylated by purified p34(cdc2) kinase. Nevertheless, we found that the properties of these RPA mutan ts were identical to those of the wild-type protein. The mutated RPA p roteins had normal single-stranded DNA binding activity and were compl etely functional for DNA replication. In addition, the mutants became hyperphosphorylated when incubated under DNA replication conditions. T hese results demonstrate that phosphorylation by p34(cdc2) kinase is n ot essential for RPA function in DNA replication in vitro. Possible ro les of RPA phosphorylation on DNA metabolism are discussed.