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| The EMBO Journal
(1997) 16, 6314–6322, doi:10.1093/emboj/16.20.6314 |
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| Opposite base-dependent reactions of a human base excision repair enzyme on DNA containing 7,8-dihydro-8-oxoguanine and abasic sites |
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| Magnar Bjørås1, Luisa Luna1, Barbro Johnsen1, Elsebeth Hoff1, Terje Haug2, Torbjørn Rognes1 and Erling Seeberg1 |
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1 Department of Molecular Biology, Institute of Medical Microbiology, University of Oslo, The National Hospital, N-0027 Oslo, Norway 2 Centre for Occupational and Environmental Medicine, The National Hospital, N-0027 Oslo, Norway To whom correspondence should be addressed Erling Seeberg, e.c.seeberg@labmed.uio.no Received 7 April 1997; Revised 4 July 1997. |
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| Abstract |
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The guanine modification 7,8-dihydro-8-oxoguanine (8-oxoG) is a potent premutagenic lesion formed spontaneously at high frequencies in the genomes of aerobic organisms. We have characterized a human DNA repair glycosylase for 8-oxoG removal, hOGH1 (human yeast OGG1 homologue), by molecular cloning and functional analysis. Expression of the human cDNA in a repair deficient mutator strain of Escherichia coli (fpg mutY) suppressed the spontaneous mutation frequency to almost normal levels. The hOGH1 enzyme was localized to the nucleus in cells transfected by constructs of hOGH1 fused to green fluorescent protein. Enzyme purification yielded a protein of 38 kDa removing both formamidopyrimidines and 8-oxoG from DNA. The enzymatic activities of hOGH1 was analysed on DNA containing single residues of 8-oxoG or abasic sites opposite each of the four normal bases in DNA. Excision of 8-oxoG opposite C was the most efficient and was followed by strand cleavage via  -elimination. However, significant removal of 8-oxoG from mispairs (8-oxoG: T >G >A) was also demonstrated, but essentially without an associated strand cleavage reaction. Assays with abasic site DNA showed that strand cleavage was indeed dependent on the presence of C in the opposite strand, irrespective of the prior removal of an 8-oxoG residue. It thus appears that strand incisions are made only if repair completion results in correct base insertion, whereas excision from mispairs preserves strand continuity and hence allows for error-free correction by a postreplicational repair mechanism. |
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| Keywords: AP-lyase, base excision, DNA glycosylase, DNA repair, 8-oxoguanine |
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