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

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Summary

Introduction

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.

Results
Conclusion

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