Gs. Brush et al., THE DNA-ACTIVATED PROTEIN-KINASE IS REQUIRED FOR THE PHOSPHORYLATION OF REPLICATION PROTEIN-A DURING SIMIAN-VIRUS-40 DNA-REPLICATION, Proceedings of the National Academy of Sciences of the United Statesof America, 91(26), 1994, pp. 12520-12524
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.