Base excision repair synthesis of DNA containing 8-oxoguanine in Escherichia coli.
8-oxo-7,8-dihydroguanine (8-oxo-G) in DNA is a mutagenic adduct formed by reactive oxygen species. In Escherichia coli, 2,6-dihydroxy-5N-formamidopyrimidine (Fapy)-DNA glycosylase (Fpg) removes this mutagenic adduct from DNA. In this report, we demonstrate base excision repair (BER) synthesis of DNA containing 8-oxo-G with Fpg in vitro. Fpg cut the oligonucleotide at the site of 8-oxo-G, producing one nucleotide gap with 3' and 5' phosphate termini. Next, 3' phosphatase(s) in the supernatant obtained by precipitating a crude extract of E. coli with 40% ammonium sulfate, removed the 3' phosphate group at the gap, thus exposing the 3' hydroxyl group to prime DNA synthesis. DNA polymerase and DNA ligase then completed the repair. These results indicate the biological significance of the glycosylase and apurinic/ apyrimidinic (AP) lyase activities of Fpg, in concert with 3' phosphatase(s) to create an appropriately gapped substrate for efficient BER synthesis of DNA containing 8-oxo-G.
Lee
Yun-Song
YS
Division of Pharmacology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea. yslee@skku.edu
Chung
Myung-Hee
MH
D045647:DNA Glycosylases / Q000378:metabolism; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry* / Q000378:metabolism*; D043382:DNA-Formamidopyrimidine Glycosylase / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics*; D006147:Guanine / Q000031:analogs & derivatives* / Q000378:metabolism*
D004269:DNA, Bacterial; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; D043382:DNA-Formamidopyrimidine Glycosylase