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Protein Engineering, Vol. 12, No. 4, 333-340, April 1999
© 1999 Oxford University Press

Methanobacterium thermoformicicum thymine DNA mismatch glycosylase: conversion of an N-glycosylase to an AP lyase

Thomas J. Begley and Richard P. Cunningham1

Department of Biological Sciences, SUNY at Albany, Albany, NY 12222, USA

The thymine DNA mismatch glycosylase from Methanobacterium thermoformicicum, a member of the endonuclease III family of repair proteins, excises the pyrimidine base from T–G and U–G mismatches. Unlike endonuclease III, it does not cleave the phosphodiester backbone by a ß-elimination reaction. This cleavage event has been attributed to a nucleophilic attack by the conserved Lys120 of endonuclease III on the aldehyde group at C1' of the deoxyribose and subsequent Schiff base formation. The inability of TDG to perform this ß-elimination event appears to be due to the presence of a tyrosine residue at the position equivalent to Lys120 in endonuclease III. The purpose of this work was to investigate the requirements for AP lyase activity. We replaced Tyr126 in TDG with a lysine residue to determine if this replacement would yield an enzyme with an associated AP lyase activity capable of removing a mismatched pyrimidine. We observed that this replacement abolishes the glycosylase activity of TDG but does not affect substrate recognition. It does, however, convert the enzyme into an AP lyase. Chemical trapping assays show that this cleavage proceeds through a Schiff base intermediate and suggest that the amino acid at position 126 interacts with C1' on the deoxyribose sugar.

Keywords: AP lyase/DNA damage/endonuclease III/thymine DNA mismatch glycosylase

1 To whom correspondence should be addressed. E-mail: moose{at}csc.albany.edu


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