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Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.

ABSTRACT: Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a mutator phenotype and are deficient in 8-oxoguanine repair. The recombinant mouse gene (mOgg1) suppresses the mutator phenotype of mutY/mutM E. coli. Extracts prepared from mutY/mutM E. coli expressing mOgg1 contain an activity that excises 8-oxoguanine from DNA and a beta-lyase activity that nicks DNA 3' to the lesion. The mouse ogg1 gene product acts efficiently on DNA duplexes in which 7, 8-dihydroxy-8-oxo-2'-deoxyguanosine (8-oxodG) is paired with dC, acts weakly on duplexes in which 8-oxodG is paired with dT or dG, and is inactive against duplexes in which 8-oxodG is paired with dA. Mouse and human ogg1 genes contain a helix-hairpin-helix structural motif with conserved residues characteristic of a recently defined family of DNA glycosylases. Ogg1 mRNA is expressed in several mouse tissues; highest levels were detected in testes. Isolation of the mouse ogg1 gene makes it possible to modulate its expression in mice and to explore the involvement of oxidative DNA damage and associated repair processes in aging and cancer.

SUBMITTER: Rosenquist TA

PROVIDER: S-EPMC23838 | biostudies-literature | 1997 Jul

REPOSITORIES: biostudies-literature

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Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.

Rosenquist T A TA Zharkov D O DO Grollman A P AP

Proceedings of the National Academy of Sciences of the United States of America 19970701 14

Oxidative DNA damage is generated by reactive oxygen species. The mutagenic base, 8-oxoguanine, formed by this process, is removed from oxidatively damaged DNA by base excision repair. Genes coding for DNA repair enzymes that recognize 8-oxoguanine have been reported in bacteria and yeast. We have identified and characterized mouse and human cDNAs encoding homologs of the 8-oxoguanine DNA glycosylase (ogg1) gene of Saccharomyces cerevisiae. Escherichia coli doubly mutant for mutM and mutY have a ...[more]

PMID: 9207108

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Project description:Elevated cellular oxidative stress and oxidative DNA damage are key contributors to impaired cardiac function in diabetes. During chronic inflammation, reactive oxygen species (ROS)-induced lipid peroxidation results in the formation of reactive aldehydes, foremost of which is 4-hydroxy-2-nonenal (4HNE). 4HNE forms covalent adducts with proteins, negatively impacting cellular protein function. During conditions of elevated oxidative stress, oxidative DNA damage such as modification by 8-hydroxydeoxyguanosine (8OHdG) is repaired by 8-oxoguanine glycosylase-1 (OGG-1). Based on these facts, we hypothesized that 4HNE forms adducts with OGG-1 inhibiting its activity, and thus, increases the levels of 8OHG in diabetic heart tissues. To test our hypothesis, we evaluated OGG-1 activity, 8OHG and 4HNE in the hearts of leptin receptor deficient db/db mice, a type-2 diabetic model. We also treated the recombinant OGG-1 with 4HNE to measure direct adduction. We found decreased OGG-1 activity (P > .05), increased 8OHG (P > .05) and increased 4HNE adducts (P > .05) along with low aldehyde dehydrogenase-2 activity (P > .05). The increased colocalization of OGG-1 and 4HNE in cardiomyocytes suggest 4HNE adduction on OGG-1. Furthermore, colocalization of 8OHG and OGG-1 with mitochondrial markers TOM 20 and aconitase, respectively, indicated significant levels of oxidatively-induced mtDNA damage and implicated a role for mitochondrial OGG-1 function. In vitro exposure of recombinant OGG-1 (rOGG-1) with increasing concentrations of 4HNE resulted in a concentration-dependent decrease in OGG-1 activity. Mass spectral analysis of trypsin digests of 4HNE-treated rOGG-1 identified 4HNE adducts on C28, C75, C163, H179, H237, C241, K249, H270, and H282. In silico molecular modeling of 4HNE-K249 OGG-1 and 4HNE-H270 OGG-1 mechanistically supported 4HNE-mediated enzymatic inhibition of OGG-1. In conclusion, these data support the hypothesis that inhibition of OGG-1 by direct modification by 4HNE contributes to decreased OGG-1 activity and increased 8OHG-modified DNA that are present in the diabetic heart.

Dataset Information

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.

Publications

Cloning and characterization of a mammalian 8-oxoguanine DNA glycosylase.

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