Cloning of a yeast 8-oxoguanine DNA glycosylase reveals the existence of a base-excision DNA-repair protein superfamily.
BACKGROUND
Reactive oxygen species, ionizing radiation, and other free radical generators initiate the conversion of guanine (G) residues in DNA to 8-oxoguanine (OG), which is highly mutagenic as it preferentially mispairs with adenine (A) during replication. Bacteria counter this threat with a multicomponent system that excises the lesion, corrects OG:A mispairs and cleanses the nucleotide precursor pool of dOGTP. Although biochemical evidence has suggested the existence of base-excision DNA repair proteins specific for OG in eukaryotes, little is known about these proteins.


RESULTS
Using substrate-mimetic affinity chromatography followed by a mechanism-based covalent trapping procedure, we have isolated a base-excision DNA repair protein from Saccharomyces cerevisiae that processes OG opposite cytosine (OG:C) but acts only weakly on OG:A. A search of the yeast genome database using peptide sequences from the protein identified a gene, OGG1, encoding a predicted 43 kDa (376 amino acid) protein, identical to one identified independently by complementation cloning. Ogg1 has OG:C-specific base-excision DNA repair activity and also intrinsic beta-lyase activity, which proceeds through a Schiff base intermediate. Targeted disruption of the OGG1 gene in yeast revealed a second OG glycosylase/lyase protein, tentatively named Ogg2, which differs from Ogg1 in that it preferentially acts on OG:G.


CONCLUSIONS
S. cerevisiae has two OG-specific glycosylase/lyases, which differ significantly in their preference for the base opposite the lesion. We suggest that one of these, Ogg1, is closely related in overall three-dimensional structure to Escherichia coli endonuclease III (endo III), a glycosylase/lyase that acts on fragmented and oxidatively damaged pyrimidines. We have recently shown that AlkA, a monofunctional DNA glycosylase that acts on alkylated bases, is structurally homologous to endo III. We have now identified a shared active site motif amongst these three proteins. Using this motif as a protein database searching tool, we find that it is present in a number of other base-excision DNA repair proteins that process diverse lesions. Thus, we propose the existence of a DNA glycosylase superfamily, members of which possess a common fold yet act upon remarkably diverse lesions, ranging from UV photoadducts to mismatches to alkylated or oxidized bases.
Nash
H M
HM
Harvard University, Department of Chemistry and Chemical Biology, 12 Oxford Street, Cambridge, Massachusetts 02138, USA.
Bruner
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SD
Schärer
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OD
Kawate
T
T
Addona
T A
TA
Spooner
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Lane
W S
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Verdine
G L
GL
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004260:DNA Repair / Q000235:genetics*; D043382:DNA-Formamidopyrimidine Glycosylase; D029968:Escherichia coli Proteins; D008969:Molecular Sequence Data; D005810:Multigene Family; D009699:N-Glycosyl Hydrolases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D009838:Oligodeoxyribonucleotides; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D013379:Substrate Specificity
D029968:Escherichia coli Proteins; D009838:Oligodeoxyribonucleotides; D009699:N-Glycosyl Hydrolases; D043382:DNA-Formamidopyrimidine Glycosylase; C468610:DNA-formamidopyrimidine glycosylase, E coli