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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

Handa, Priya and Roy, Sudipta and Varshney, Umesh (2001) 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. In: Journal of Biological Chemistry, 276 (20). pp. 17324-17331.

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Abstract

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 $L_{191}$ within the $_{187}\hspace{1mm}HPSPLS_{192}$ 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 L191 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 L191 in stabilizing the productive enzyme-substrate complex.

Item Type: Journal Article
Additional Information: Copyright of this article belongs to American Society for Biochemistry and Molecular Biology.
Department/Centre: Division of Biological Sciences > Microbiology & Cell Biology
Date Deposited: 04 Oct 2007
Last Modified: 19 Sep 2010 04:40
URI: http://eprints.iisc.ernet.in/id/eprint/12058

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