The C-terminal domain of the adenine-DNA glycosylase MutY confers specificity for 8-oxoguanine.adenine mispairs and may have evolved from MutT, an 8-oxo-dGTPase.
MutY is an adenine-DNA glycosylase with specificity for mismatches involving 8-oxoguanine (oG.A) or guanine (G.A). In addition to a 25 kDa catalytic domain common to all members of its DNA glycosylase superfamily, MutY has a 14 kDa C-terminal domain. Sequence analyses suggest that this C-terminal domain is distantly related to MutT, a pyrophosphohydrolase specific for 2'-deoxy-8-oxoguanosine triphosphate (doGTP). Here we present biochemical evidence that the MutT-like domain of MutY is the principal determinant of oG specificity. First, MutY dissociates approximately 1500-fold more slowly from oG-containing product DNA than from G-containing product, but a truncated protein lacking the C-terminal domain dissociates as rapidly from oG-DNA as the full-length protein dissociates from G-DNA. Second, MutY removes adenine from oG.A mismatches almost 30-fold faster than from G.A mismatches in a pre-steady-state assay, but deletion of the C-terminal domain reduces this specificity for oG.A to less than 4-fold. The kinetic data are consistent with a model in which binding of oG to the C-terminal domain of MutY accelerates the pre-steady-state glycosylase reaction by facilitating adenine base flipping. The observation that oG specificity derives almost exclusively from the C-terminal domain of MutY adds credence to the sequence analyses and suggests that specificity for oG.A mismatches was acquired by fusion of a MutT-like protein onto the core catalytic domain of an adenine-DNA glycosylase.
Noll
D M
DM
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Baltimore, Maryland 21205, USA.
Gogos
A
A
Granek
J A
JA
Clarke
N D
ND
D000225:Adenine / Q000737:chemistry* / Q000378:metabolism; D000595:Amino Acid Sequence; D020137:Base Pair Mismatch; D045647:DNA Glycosylases; D004260:DNA Repair; D004269:DNA, Bacterial / Q000737:chemistry / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology; D006147:Guanine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid
D004269:DNA, Bacterial; D010446:Peptide Fragments; C024829:8-hydroxyguanine; D006147:Guanine; D045647:DNA Glycosylases; D009699:N-Glycosyl Hydrolases; C061738:mutY adenine glycosylase; D000225:Adenine