Flexibility at Gly-194 Is Required for DNA Cleavage and Relaxation Activity of Escherichia coli DNA Topoisomerase I

Abstract

The proposed mechanism of type IA DNA topoisomerase I includes conformational changes by the single enzyme polypeptide to allow binding of the G strand of the DNA substrate at the active site, and the opening or closing of the "gate" created on the G strand of DNA to the passing single or double DNA strand(s) through the cleaved G strand DNA. The shifting of an α helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures (Changela, A., DiGate, R. J., and Mondragón, A. (2001) Nature 411, 1077-1081). Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this a helix in Escherichia coli DNA topoisomerase I showed that flexibility around this glycine residue is required for DNA cleavage and relaxation activity and supports a functional role for this hinge region in the enzyme conformational change.