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Journal of Biochemistry Advance Access originally published online on June 24, 2009
Journal of Biochemistry 2009 146(4):523-526; doi:10.1093/jb/mvp095
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© The Authors 2009. Published by Oxford University Press on behalf of the Japanese Biochemical Society. All rights reserved

Changes in the Conformation of the Vsr Endonuclease Amino-terminal Domain Accompany DNA Cleavage

Yaroslava Y. Polosina* and Claire G. Cupples

Department of Biochemistry and Microbiology, University of Victoria, PO Box 3055 STN CSC, Victoria, BC, V8W 3P6, Canada

*To whom correspondence should be addressed. Tel: +250-721-8883, Fax: +250-721-8855, E-mail: polosina{at}uvic.ca

Received March 16, 2009; Accepted June 15, 2009


  Abstract

In Escherichia coli, T/G mismatches arising from deamination of 5-methylcytosine to thymine are converted to CG base pairs by the very short patch (VSP) repair pathway. DNA Polymerase I removes and resynthesizes the mismatched T starting from a 5'-nick created by the Vsr endonuclease. We used limited trypsinolysis to probe conformational changes in the N-terminal domain of Vsr in response to DNA binding, DNA cleavage and interaction with the polymerase. Our data show that the domain becomes trypsin resistant only under conditions that allow DNA cleavage, while interaction with the polymerase restores trypsin sensitivity. We suggest that the domain changes its conformation as a result of DNA nicking, and that DNA Pol I releases Vsr from the nick by reversing that conformational change.

Key Words: DNA polymerase I, DNA repair, limited tryptic digestion, VSP repair, Vsr endonuclease

Abbreviations: LB, Luria-Bertani medium; IPTG, isopropyl-β-D-thiogalactopyranoside; SSC, saline-medium citrate buffer; SDS, sodium dodecyl sulphate; LC-MS, Liquid chromatography-mass spectrometry


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