Requirement for PCNA and RPA in interstrand crosslink-induced DNA synthesis

Proliferating nuclear cell antigen (PCNA) and replication protein A (RPA) h ave proven to be essential elements in many aspects of DNA metabolism inclu ding replication, repair and recombination. We have developed an in vitro a ssay in which the presence of an interstrand crosslink stimulates the incor poration of radiolabeled nucleotides into both damaged and undamaged plasmi d DNAs. Using this assay we have investigated the roles of PCNA and RPA in crosslink-induced DNA synthesis. p21, a potent inhibitor of PCNA, was found to strongly inhibit crosslink-induced incorporation. Addition of exogenous PCNA partially restored the resynthesis activity. Likewise, neutralization of RPA by monoclonal antibodies also inhibited incorporation, but the effe ct was somewhat more pronounced on the undamaged plasmid than the damaged p lasmid, Addition of excess RPA also partially reversed antibody inhibition. These results indicate that both PCNA and RPA are required for efficient i n vitro DNA resynthesis induced by interstrand crosslinks.