Nucleic Acids Research, Vol 26, Issue 22 5123-5133, Copyright © 1998 by Oxford University Press
SD Williams and SS David
The Escherichia coli adenine glycosylase MutY is involved in the repair of
7,8-dihydro-8-oxo-2'-deoxyguanosine (OG):A and G:A mispairs in DNA. DNA
strand cleavage via beta-elimination (beta-lyase) activity coupled with
MutY's removal of misincorporated adenine bases was sought using both
qualitative and quantitative methods. The qualitative assays demonstrate
formation of a Schiff base intermediate which is characteristic of DNA
glycosylases catalyzing a concomitant beta-lyase reaction. Borohydride
reduction of the Schiff base results in the formation of a covalent
DNA-MutY adduct which is easily detected in SDS- PAGE experiments. However,
quantitative activity assays which monitor DNA strand scission accompanying
base release suggest MutY behaves as a simple monofunctional glycosylase.
Treatment with base effects DNA strand cleavage at apurinic/apyrimidinic
(AP) sites arising via simple glycosylase activity. The amount of cleaved
DNA in MutY reactions treated with base is much greater than that in
non-base treated reactions, indicating that AP site generation by MutY is
not associated with a concomitant beta-lyase step. As standards, identical
assays were performed with a known monofunctional enzyme (uracil DNA
glycosylase) and a known bifunctional glycosylase/lyase (FPG), the results
of which were used in comparison with those of the MutY experiments. The
apparent inconsistency between the data obtained for MutY by the
qualitative and quantitative methods underscores the current debate
surrounding the catalytic activity of this enzyme, and a detailed
explanation of this controversy is proposed. The work presented here lays
ground for the identification of specific active site residues responsible
for the chemical mechanism of MutY enzyme catalysis.
ARTICLES
Evidence that MutY is a monofunctional glycosylase capable of forming a covalent Schiff base intermediate with substrate DNA
Department of Chemistry, University of Utah, Salt Lake City, UT 84112, USA.
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