La. Henricksen et Ms. Wold, REPLICATION PROTEIN-A MUTANTS LACKING PHOSPHORYLATION SITES FOR P34(CDC2) KINASE SUPPORT DNA-REPLICATION, The Journal of biological chemistry, 269(39), 1994, pp. 24203-24208
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.