Roles of replication protein a and DNA-dependent protein kinase in the regulation of DNA replication following DNA damage

Exposure of mammalian cells to DNA damage-inducing agents (DDIA) inhibits o ngoing DNA replication. The molecular mechanism of this inhibition remains to be elucidated. We employed a simian virus 40 (SV40) based in vitro DNA r eplication assay to study biochemical aspects of this inhibition. We report here that the reduced DNA replication activity in extracts of DDIA-treated cells is partly caused by a reduction in the amount of replication protein A (RPA). We also report that the dominant inhibitory effect is caused by t he DNA-dependent protein kinase (DNA-PK) which inactivates SV40 T antigen ( TAg) by phosphorylation. The results demonstrate that RPA and DNA-PK are in volved in the regulation of viral DNA replication after DNA damage and sugg est that analogous processes regulate cellular DNA replication with the DNA -PK targeting the functional homologues of TAg.