Specific recognition of A/G and A/7,8-dihydro-8-oxoguanine (8-oxoG) mismatches by Escherichia coli MutY: removal of the C-terminal domain preferentially affects A/8-oxoG recognition.
Escherichia coli MutY is a 39 kDa adenine DNA glycosylase and 3' apurinic/apyrimidinic (AP) lyase that is active on DNA substrates containing A/G, A/C, or A/8-oxoG mismatches. 8-oxoG (7,8-dihydro-8-oxoguanine or GO) is a major stable product of oxidative damage, and A/GO mismatches may be particularly important biological substrates for MutY. Proteolytic digestion of MutY using thermolysin was found to produce two relatively stable fragments of 25 and 12 kDa. The 25 kDa fragment begins at the N terminus of MutY and spans the region homologous with E. coli endonuclease III, a DNA glycosylase/AP lyase that repairs oxidatively damaged pyrimidines. The 12 kDa fragment, which consists of much of the rest of MutY, had no detectable activity. The purified 25 kDa fragment (M25) had nearly wild-type binding and cleavage activities with A/G-mismatched substrates. Binding to A/GO-mismatched DNA, however, was dramatically reduced in M25 compared to that in intact protein. Borohydride-dependent enzyme-DNA cross-linking, which is a hallmark of the reaction of several DNA glycosylases that possess concomitant AP lyase activity, was also substantially reduced when M25 was allowed to react with A/GO-mismatched DNA. The significant differences in M25 recognition and reactivity with A/G and A/GO mismatches suggest that the C-terminal region of MutY, a region with no homologous counterpart in E. coli endonuclease III, plays an important role in the repair of mismatched DNA arising from oxidation damage.
Gogos
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Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Cillo
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Clarke
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Lu
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D000225:Adenine / Q000737:chemistry; D000595:Amino Acid Sequence; D001482:Base Composition; D001483:Base Sequence; D001665:Binding Sites; D004249:DNA Damage; D045647:DNA Glycosylases; D004260:DNA Repair; D010450:Endopeptidases; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D006147:Guanine / Q000031:analogs & derivatives / Q000737:chemistry; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D009838:Oligodeoxyribonucleotides / Q000737:chemistry / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D010446:Peptide Fragments; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D010450:Endopeptidases; D000225:Adenine