Replication Protein A Stimulates Proliferating Cell Nuclear Antigen-Dependent Repair of Abasic Sites in DNA by Human Cell Extracts

Abstract

Base excision repair (BER) pathway is the major cellular process for removal of endogenous base lesions and apurinic/apyrimidinic (AP) sites in DNA. There are two base excision repair subpathways in mammalian cells, characterized by the number of nucleotides synthesized into the excision patch. They are the “single-nucleotide” (one nucleotide incorporated) and the “long-patch” (several nucleotides incorporated) BER pathways. Proliferating cell nuclear 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) in PCNA-dependent, long-patch BER of AP sites in human cell extracts. PCNA and RPA were separated from the other BER proteins by fractionation of human whole-cell extract on a phosphocellulose column. The protein fraction PC-FII (phosphocellulose fraction II), which does not contain RPA and PCNA but otherwise contains all core BER proteins required for PCNA-dependent BER (AP endonuclease, DNA polymerases δ, β and DNA ligase, and FEN1 endonuclease), had reduced ability to repair plasmid DNA containing AP sites. Purified PCNA or RPA, when added separately, could only partially restore the PC-FII repair activity of AP sites. However, additions of both proteins together greatly stimulated AP site repair by PC-FII. These results demonstrate a role for RPA in PCNA-dependent BER of AP sites.