Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase

The human protein MED1 (also known as MBD4) was previously isolated in a tw o-hybrid screening using the mismatch repair protein MLH1 as a bait, and sh own to have homology to bacterial base excision repair DNA N-glycosylases/l yases. To define the mechanisms of action of MED1, we implemented a sensiti ve glycosylase assay amenable to kinetic analysis. We show that MED1 functi ons as a mismatch-specific DNA N-glycosylase active on thymine, uracil, and 5-fluorouracil when these bases are opposite to guanine, MED1 lacks uracil glycosylase activity on single-strand DNA and abasic site lyase activity. The glycosylase activity of MED1 prefers substrates containing a G:T mismat ch within methylated or unmethylated CpG sites; since G:T mismatches can or iginate via deamination of 5-methylcytosine to thymine, MED1 may act as a c aretaker of genomic fidelity at CpG sites. A kinetic analysis revealed that MED1 displays a fast first cleavage reaction followed by slower subsequent reactions, resulting in biphasic time course; this is due to the tight bin ding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the ME D1 5-methylcytosine binding domain and methylation of the mismatched CpG si te are not required for efficient catalysis.