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

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.

The cutting edges in DNA repair, licensing, and fidelity: DNA and RNA repair nucleases sculpt DNA to measure twice, cut once

“…Unfortunately, the rate of product release has not been measured. Interestingly, the N-terminal helix appears not to clamp down until the product has been formed, as incubation with Mg 2+ but not Ca 2+ protected it from limited proteolysis [91]. This suggests that protection of the product DNA was engineered into the Vsr N-terminus.…”