Biphasic Kinetics of the Human DNA Repair Protein MED1 (MBD4), a Mismatch-specific DNA N-Glycosylase

• Published in: The Journal of biological chemistry Vol. 275; no. 42; pp. 32422 - 32429
• Main Authors: Petronzelli, Fiorella, Riccio, Antonio, Markham, George D., Seeholzer, Steven H., Stoerker, Jay, Genuardi, Maurizio, Yeung, Anthony T., Matsumoto, Yoshihiro, Bellacosa, Alfonso
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 20.10.2000; American Society for Biochemistry and Molecular Biology
• Subjects: Base Pair Mismatch; Base Sequence; DNA Repair; Endodeoxyribonucleases - genetics; Endodeoxyribonucleases - metabolism; Humans; Kinetics; MBD4 protein; MED1 protein; Molecular Sequence Data; Mutagenesis; Oligodeoxyribonucleotides - chemical synthesis; Oligodeoxyribonucleotides - chemistry; Oligodeoxyribonucleotides - metabolism; Recombinant Proteins - metabolism; Sensitivity and Specificity; Sequence Deletion; Substrate Specificity
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M004535200

The 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.