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 alpha helix upon G strand DNA binding has been observed from the comparison of the type IA DNA topoisomerase crystal structures. Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this alpha 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.
Highlights
DNA topoisomerases are ubiquitous enzymes involved in DNA replication, transcription, and recombination
The G194R and G194A mutant enzymes expressed in GP200 were purified to Ͼ95% homogeneity with procedures similar to those used for the wild-type topoisomerase I and assayed for relaxation activity (Fig. 3)
A number of conserved amino acids around the active site of E. coli DNA topoisomerase I have been shown by site-directed mutagenesis studies (18 –20) to be involved in the DNA cleavage step of catalysis by the enzyme
Summary
DNA topoisomerases are ubiquitous enzymes involved in DNA replication, transcription, and recombination (reviewed in Refs. 1–3). Site-directed mutagenesis of the strictly conserved Gly-194 at the N terminus of this ␣ 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.
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