THE DNA-ACTIVATED PROTEIN-KINASE IS REQUIRED FOR THE PHOSPHORYLATION OF REPLICATION PROTEIN-A DURING SIMIAN-VIRUS-40 DNA-REPLICATION

The 32-kDa subunit of replication protein A (RPA) is phosphorylated du ring the S phase of the cell cycle in vivo and during simian virus 40 DNA replication in vitro. To explore the functional significance of th is modification, we purified a HeLa fell protein kinase that phosphory lates RPA in the presence of single-stranded DNA. By several criteria we identified the purified enzyme as a form of the DNA-activated prote in kinase (DNA-PK), a previously described high molecular weight prote in kinase that is capable of phosphorylating a number of nuclear DNA b inding proteins. Phosphorylation of RPA by DNA-PR is stimulated by nat ural single-stranded DNAs but not by homopolymers lacking secondary st ructure. Studies with the simian virus 40 model system indicate that D NA-PK is required for DNA-replication-dependent RPA phosphorylation. D epletion of the kinase activity, however, has no effect on the extent of DNA replication in vitro. Our data support a model in which phospho rylation of RPA by DNA-Pk is activated by formation of replication int ermediates containing single- and double-stranded regions. This event may be involved in a signaling mechanism that coordinates DNA replicat ion with the cell cycle.