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J. Biol. Chem., Vol. 276, Issue 20, 17324-17331, May 18, 2001
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From the Department of Microbiology and Cell Biology, Indian
Institute of Science, Bangalore, 560 012, India
Uracil DNA glycosylase (UDG), a
highly conserved DNA repair enzyme, initiates the uracil excision
repair pathway. Ugi, a bacteriophage-encoded peptide, potently inhibits
UDGs by serving as a remarkable substrate mimic. Structure
determination of UDGs has identified regions important for the
exquisite specificity in the detection and removal of uracils from DNA
and in their interaction with Ugi. In this study, we carried out
mutational analysis of the Escherichia coli UDG at
Leu191 within the
187HPSPLS192 motif (DNA intercalation loop). We
show that with the decrease in side chain length at position 191, the
stability of the UDG-Ugi complexes regresses. Further, while the L191V
and L191F mutants were as efficient as the wild type protein, the L191A
and L191G mutants retained only 10 and 1% of the enzymatic activity,
respectively. Importantly, however, substitution of Leu191
with smaller side chains had no effect on the relative efficiencies of
uracil excision from the single-stranded and a corresponding double-stranded substrate. Our results suggest that leucine within the
HPSPLS motif is crucial for the uracil excision activity of UDG, and it
contributes to the formation of a physiologically irreversible complex
with Ugi. We also envisage a role for Leu191 in stabilizing
the productive enzyme-substrate complex.
The Role of Leucine 191 of Escherichia coli
Uracil DNA Glycosylase in the Formation of a Highly Stable Complex with
the Substrate Mimic, Ugi, and in Uracil Excision from the Synthetic
Substrates*
,
*
This work was supported in part by research grants from the
Council of Scientific and Industrial Research (CSIR), the Department of
Biotechnology, and the Department of Science and Technology, New Delhi,
India.The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
Supported by a senior research fellowship of CSIR.
§
To whom correspondence should be addressed. Tel.:
91-80-309-2686; Fax: 91-80-360-2697 or 91-80-360-0683; E-mail:
varshney@ mcbl.iisc.ernet.in.
Copyright © 2001 by The American Society for Biochemistry and Molecular Biology, Inc.
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