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Originally published In Press as doi:10.1074/jbc.M011166200 on February 8, 2001

J. Biol. Chem., Vol. 276, Issue 20, 17324-17331, May 18, 2001
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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*

Priya HandaDagger , Sudipta Roy, and Umesh Varshney§

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.

* 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.

Dagger 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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