Phosphorylation of replication protein a middle subunit (RPA32) leads to adisassembly of the RPA heterotrimer

Replication protein A (RPA), the major eukaryotic single-strand specific DN A binding protein, consists of three subunits, RPA70, RPA32, and RPA14. The middle subunit, RPA32, is phosphorylated in a cell cycle-dependent manner. RPA occurs in two nuclear compartments, bound to chromatin or free in the nucleosol, We show here that the chromatin-associated fraction of RPA conta ins the phosphorylated forms of RPA32, Treatment of chromatin with 0.4 hr N aCl releases bound RPA and causes a separation of the large and the phospho rylated middle RPA subunit, Unmodified RPA in the nucleosolic fraction rema ins perfectly stable under identical conditions. Phosphorylation is most li kely an important determinant of RPA desintegration because dialysis from 0 .4 to 0.1 NaCl causes the reformation of trimeric RPA only under dephosphor ylating conditions, Biochemical studies with isolated Cyclin-dependent prot ein kinases showed that cyclin A/CDK1 and cyclin B/CDK1, but not cyclin E/C DK2, can phosphorylate human recombinant RPA in vitro, However, only a smal l fraction of in vitro phosphorylated RPA desintegrated, suggesting that ph osphorylation may be one, but probably not the only, determinant affecting subunit interaction. We speculate that phosphorylation and changes in subun it interaction are required for the proposed role of RPA during the polymer ase switch at replication forks.