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 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 delta, beta 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.
Dianov
G L
GL
Laboratory of Molecular Genetics, National Institute on Aging, National Institutes of Health, Baltimore, Maryland 21224, USA. dianovg@grc.nia.nih.gov
Jensen
B R
BR
Kenny
M K
MK
Bohr
V A
VA
D001483:Base Sequence; D019757:Carbon-Oxygen Lyases / Q000378:metabolism*; D002411:Cation Exchange Resins; D002458:Cell Fractionation; D002482:Cellulose / Q000031:analogs & derivatives; D002850:Chromatography, Gel; D004247:DNA / Q000378:metabolism*; D004260:DNA Repair; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000502:physiology*; D043223:Deoxyribonuclease IV (Phage T4-Induced); D004357:Drug Synergism; D006801:Humans; D008214:Lymphocytes / Q000201:enzymology / Q000378:metabolism*; D008969:Molecular Sequence Data; D018809:Proliferating Cell Nuclear Antigen / Q000302:isolation & purification / Q000502:physiology*; D051716:Replication Protein A
D002411:Cation Exchange Resins; D004268:DNA-Binding Proteins; D018809:Proliferating Cell Nuclear Antigen; C497167:RPA1 protein, human; D051716:Replication Protein A; D002482:Cellulose; D004247:DNA; C008881:phosphocellulose; D043223:Deoxyribonuclease IV (Phage T4-Induced); D019757:Carbon-Oxygen Lyases; D043603:DNA-(Apurinic or Apyrimidinic Site) Lyase