Abstract
5-Hydroxy-2'-deoxycytidine (5-OHdC) and 5-hydroxy-2'-deoxyuridine (5-OHdU) are major products of oxidative DNA damage with mutagenic potential. Until now, no enzymatic activity responsible for their removal has been identified. We report here that both 5-OHdC and 5-OHdU are substrates for Escherichia coli endonuclease III and formamidopyrimidine DNA N-glycosylase (FPG). 5-OHdU is also a substrate for uracil DNA N-glycosylase. Consistent with their mechanisms of action on previously described substrates, endonuclease III removes 5-OHdC and 5-OHdU via a N-glycosylase/beta-elimination reaction, FPG follows a N-glycosylase/beta,delta-elimination reaction, and uracil N-glycosylase removes 5-OHdU by N-glycosylase action leaving behind an abasic site. Endonuclease III removes both lesions more efficiently than FPG, and both endonuclease III and FPG remove 5-OHdC slightly more efficiently than 5-OHdU. Uracil DNA N-glycosylase removes 5-OHdU more efficiently than the other two enzymes and has no activity on 5-OHdC even when present in great excess. Analysis of crude extracts obtained from wild type and endonuclease III deletion mutants of E. coli correlated well with data obtained with the purified enzymes.
Highlights
5-HYDROXY-2”DEOXYCYIDINAEND 5-HYDROXY-2”DEOXYURIDINAERE SUBSTRATES FOR ESCHERICHIA COLI ENDONUCLEASE I11 AND FORMAMIDOPYRIMIDINE DNA N-GLYCOSYLASE,WHILE 5-HYDROXY-2”DEOXYURIDINEISA SUBSTRATE FOR URACIL DNA N-GLYCOSYLASE*
5-OHdU byN-glycosylase action leaving behind an abaincluding endonucleases 111, IV and VIII, exonuclease 111, formamidopyrimidine DNA N-glycosylase (FPG),’ anduracil DNA N-glycosylase (UNG)
All of the above strongly suggests that these lesions are pre-mutagenic and that mechanisms must exist toremove them from DNA. In this reporwt e present data showing the removal of 5-OHdC and 5-OHdU by three different E. coli base excision repair enzymes, which were not shown previously to share common substrates contain8inMgurea in90 mM Tris-HC1,gO mM boric acid, pH 8.3,2.m5M EDTA
Summary
Sequence context of two 45 nucleotide substrates Ollgonucleotlde 1 5' GGCGGCCCCC - W 3'CCGCCGGCGAGAmmcm-cmmAAG. Double stranded DNA substrate with single site-specific lesion (sequence context Is ldentlcrl l o that of ollgonucleotlde I). 2. Extend wth Sequenare and natural dNTPs. strand during in vitro synthesis by DNA polymerase I, insertion of both dGand dAopposite 5-OHdC was observed (dG>dA), and dA was exclusively inserted opposite 5-OHdU. All of the above strongly suggests that these lesions are pre-mutagenic and that mechanisms must exist toremove them from DNA. In this reporwt e present data showing the removal of 5-OHdC and 5-OHdU by three different E. coli base excision repair enzymes, which were not shown previously to share common substrates (with theexception of rylamide, obtained from National Diagnostics) contain8inMgurea in mM Tris-HC1,gO mM boric acid, pH 8.3,2.m5M EDTA. The concentration of the acrylamide varied between 8 and 15% dependingon the experiment.Gelswereelectrophoresed for 1.5-2 h at 1800-2500 V, dried undervacuum,and exposed to x-rayfilm.Alternatively, a Bio-Rad model GS-250 molecular imager system was usedfor detection
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