Replication protein A stimulates proliferating cell nuclear antigen-dependent repair of abasic sites in DNA by human cell extracts

Base excision repair (BER) pathway is the major cellular process for remova l of endogenous base lesions and apurinic/apyrimidinic (AP) sites in DNA. T here are two base excision repair subpathways in mammalian cells, character ized by the number of nucleotides synthesized into the excision patch. They are the "single-nucleotide'' (one nucleotide incorporated) and the "long-p atch" (several nucleotides incorporated) BER pathways. Proliferating cell n uclear antigen (PCNA) is known to be an essential factor in long-patch base excision repair. We have studied the role of replication protein A (RPA) i n PCNA-dependent, long-patch BER of AP sites in human cell extracts. PCNA a nd RPA were separated from the other BER proteins by fractionation of human whole-cell extract on a phosphocellulose column. The protein fraction PC-F II (phosphocellulose fraction II), which does not contain RPA and PCNA but otherwise contains all core BER proteins required for PCNA-dependent EER (A P endonuclease, DNA polymerases delta, beta and DNA ligase, and FEN1 endonu clease), had reduced ability to repair plasmid DNA containing AP sites. Pur ified PCNA or RPA, when added separately, could only partially restore the PC-FII repair activity of AP sites. However, additions of both proteins tog ether greatly stimulated AP site repair by PC-FII. These results demonstrat e a role for RPA in PCNA-dependent EER of AP sites.