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Petronzelli F , Riccio A , Markham GD , Seeholzer SH , Stoerker J , Genuardi M , Yeung AT , Matsumoto Y , Bellacosa A
Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase
J Biol Chem. 2000 Oct 20;275(42) :32422-9
AbstractThe human protein MED1 (also known as MBD4) was previously isolated in a two-hybrid screening using the mismatch repair protein MLH1 as a bait, and shown to have homology to bacterial base excision repair DNA N-glycosylases/lyases. To define the mechanisms of action of MED1, we implemented a sensitive glycosylase assay amenable to kinetic analysis. We show that MED1 functions 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 mismatch within methylated or unmethylated CpG sites; since G:T mismatches can originate via deamination of 5-methylcytosine to thymine, MED1 may act as a caretaker 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 binding of MED1 to the abasic site reaction product rather than a consequence of enzyme inactivation. Comparison of kinetic profiles revealed that the MED1 5-methylcytosine binding domain and methylation of the mismatched CpG site are not required for efficient catalysis.
NotesPetronzelli, F Riccio, A Markham, G D Seeholzer, S H Stoerker, J Genuardi, M Yeung, A T Matsumoto, Y Bellacosa, A CA06927/CA/NCI NIH HHS/United States CA71426/CA/NCI NIH HHS/United States CA78412/CA/NCI NIH HHS/United States Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S. United states The Journal of biological chemistry J Biol Chem. 2000 Oct 20;275(42):32422-9.