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 (degrees G.A) or guanine (G.A). Ln 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 (d degrees GTP). Here we present biochemical evidence that the MutT-like domain of MutY is the principal determinant of degrees G specificity. First, MutY dissociates approximately 1500-fold more slowly from degrees G-containing product DNA than from G-containing product, but a truncated protein lacking the C-terminal domain dissociates as rapidly from degrees G-DNA as the full-length protein dissociates from G-DNA. Second, MutY removes adenine from degrees G 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 degrees G.A to less than 4-fold. The kinetic data are consistent with a model in which binding of degrees G to the C-terminal domain of MutY accelerates the pre-steady-state glycosylase reaction by facilitating adenine base flipping. The observation that degrees G specificity derives almost exclusively from the C-terminal domain of MutY adds credence to the sequence analyses and suggests that specificity for degrees G.A mismatches was acquired by fusion of a MutT-like protein onto the core catalytic domain of an adenine-DNA glycosylase.