Phosphorylation of replication protein A middle subunit (RPA32) leads to a disassembly of the RPA heterotrimer.
Replication protein A (RPA), the major eukaryotic single-strand specific DNA 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 contains the phosphorylated forms of RPA32. Treatment of chromatin with 0.4 M NaCl releases bound RPA and causes a separation of the large and the phosphorylated middle RPA subunit. Unmodified RPA in the nucleosolic fraction remains perfectly stable under identical conditions. Phosphorylation is most likely an important determinant of RPA desintegration because dialysis from 0.4 to 0.1 NaCl causes the reformation of trimeric RPA only under dephosphorylating conditions. Biochemical studies with isolated Cyclin-dependent protein kinases showed that cyclin A/CDK1 and cyclin B/CDK1, but not cyclin E/CDK2, can phosphorylate human recombinant RPA in vitro. However, only a small fraction of in vitro phosphorylated RPA desintegrated, suggesting that phosphorylation may be one, but probably not the only, determinant affecting subunit interaction. We speculate that phosphorylation and changes in subunit interaction are required for the proposed role of RPA during the polymerase switch at replication forks.
Treuner
K
K
Department of Biology, Universität Konstanz, D-78457 Konstanz, Germany.
Findeisen
M
M
Strausfeld
U
U
Knippers
R
R
D016203:CDC2 Protein Kinase / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D002843:Chromatin / Q000378:metabolism; D004261:DNA Replication / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry*; D006367:HeLa Cells; D006801:Humans; D019169:Jurkat Cells; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry; D051716:Replication Protein A; D012965:Sodium Chloride / Q000494:pharmacology
D002843:Chromatin; D004268:DNA-Binding Proteins; C497167:RPA1 protein, human; D011994:Recombinant Proteins; D051716:Replication Protein A; D012965:Sodium Chloride; D016203:CDC2 Protein Kinase