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. 1997 Jul 22;94(15):8016-20.
doi: 10.1073/pnas.94.15.8016.

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

T Roldán-Arjona  1 , Y F Wei, K C Carter, A Klungland, C Anselmino, R P Wang, M Augustus, T Lindahl
Affiliations

Molecular cloning and functional expression of a human cDNA encoding the antimutator enzyme 8-hydroxyguanine-DNA glycosylase

T Roldán-Arjona et al. Proc Natl Acad Sci U S A. .

Abstract

The major mutagenic base lesion in DNA caused by exposure to reactive oxygen species is 8-hydroxyguanine (8-oxo-7, 8-dihydroguanine). In bacteria and Saccharomyces cerevisiae, this damaged base is excised by a DNA glycosylase with an associated lyase activity for chain cleavage. We have cloned, sequenced, and expressed a human cDNA with partial sequence homology to the relevant yeast gene. The encoded 47-kDa human enzyme releases free 8-hydroxyguanine from oxidized DNA and introduces a chain break in a double-stranded oligonucleotide specifically at an 8-hydroxyguanine residue base paired with cytosine. Expression of the human protein in a DNA repair-deficient E. coli mutM mutY strain partly suppresses its spontaneous mutator phenotype. The gene encoding the human enzyme maps to chromosome 3p25. These results show that human cells have an enzyme that can initiate base excision repair at mutagenic DNA lesions caused by active oxygen.

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Figures

Figure 1
Figure 1
Sequence homology between human 8-oxoG-DNA glycosylase and S. cerevisiae OGG1. Amino acid sequences were aligned by the pileup and bestfit programs in the Genetics Computer Group package (Version 7; ref. 20). Amino acids identical in both sequences are boxed. The position of the possible helix–hairpin–helix motif is indicated. The Lys and Asp residues that have been associated with dual DNA glycosylase/AP lyase activity (13) are shown with an arrow and a circle, respectively.
Figure 2
Figure 2
Purification of recombinant hOGG1 protein. The hOGG1 was overexpressed in E. coli, and proteins were visualized on a 10% SDS/polyacrylamide gel by Coomassie blue staining. Lanes 1–4 show overexpression of the protein at 37°C by IPTG induction and contain whole-cell lysate (5 μl) from bacteria harboring the vector before (lane 1) and after induction (lane 2), or the plasmid carrying hOGG1 cDNA before (lane 3) and after induction (lane 4). Lanes 5–10 show peak fractions (10 μl) eluted with 0.5 M imidazole from a Ni2+-nitrilotriacetic acid column loaded with a crude extract from cells grown at 15°C (see Materials and Methods). The few protein bands migrating below hOGG1 represent degradation or premature termination products of the overexpressed protein, as determined by immunoblotting with the T7⋅Tag Antibody (data not shown). Positions of protein size markers in kDa (Bio-Rad) are indicated on the left.
Figure 3
Figure 3
Comparison of hOGG1 activity on double-stranded oligonucleotides containing an 8-oxoG⋅C or an 8-oxoG⋅A mismatch. Oligonucleotides were 32P-labeled at the 5′ end of the 8oxoG-containing strand and were incubated at 37°C for 1 h with purified hOGG1 as described in Materials and Methods. Reaction products were analyzed by autoradiography after electrophoretic separation in a denaturing 20% polyacrylamide gel.
Figure 4
Figure 4
Antimutator effect of hOGG1 cDNA in E. coli (mutM mutY). AB1157 (wild type) or MK611 (mutM mutY) cells transformed with pSE420 (open bars) or pSE420 carrying the hOGG1 cDNA (solid bars) were grown in the presence of 1 mM IPTG and plated both in the presence and absence of rifampicin. Results are from five independent experiments, and error bars are shown.
Figure 5
Figure 5
Fluorescence in situ hybridization mapping of the hOGG1 gene. The hOGG1 cDNA was hybridized to normal human male chromosomes. (A) A chromosome spread from a single cell, showing hybridization to the p arm of each chromosome 3 (arrows). (B) Example of a single chromosome 3 with hybridization signal. Background signals in nonchromosomal areas were suppressed in the electronic image for both A and B. (C) Idiogram of chromosome 3, showing the position of the hOGG1 gene.
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