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.

Petronzelli, F., Riccio, A., Markham, G., Seeholzer, S., Stoerker, J., Genuardi, M., Yeung, A., Matsumoto, Y., Bellacosa, A., Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase, <<THE JOURNAL OF BIOLOGICAL CHEMISTRY>>, 2000; 275 (42): 32422-32429. [doi:10.1074/jbc.M004535200]

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

Genuardi, M;
2000

Abstract

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.
Inglese
Petronzelli, F., Riccio, A., Markham, G., Seeholzer, S., Stoerker, J., Genuardi, M., Yeung, A., Matsumoto, Y., Bellacosa, A., Biphasic kinetics of the human DNA repair protein MED1 (MBD4), a mismatch-specific DNA N-glycosylase, <<THE JOURNAL OF BIOLOGICAL CHEMISTRY>>, 2000; 275 (42): 32422-32429. [doi:10.1074/jbc.M004535200]
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Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/10807/219900
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