Bacterial type IA DNA topoisomerase as a target for novel antibacterial agents

Abstract

At least one type IA DNA topoisomerases can be found in every bacterium, making it a logical target for antibacterial agents that can convert the enzyme into poison by trapping its covalent complex with DNA. We isolated a mutant of recombinant Yersinia pestis topoisomerase I by screening for its ability to induce the SOS response in E. coli. Overexpression of this mutant topoisomerase I results in bacterial cell death. From sequence analysis and site-directed mutagenesis, it was determined that a single amino acid substitution in the TOPRIM domain changing a strictly conserved glycine residue to serine in either the Y. pestis or Escherichia coli topoisomerase I can result in a mutant enzyme that has the SOS inducing and cell killing properties. Analysis of the purified mutant enzymes showed that they have no relaxation activity but retain the ability to cleave DNA and form a covalent complex. These results demonstrate that perturbation of the active site region of bacterial topoisomerase I can result in stabilization of the covalent intermediate, with the in vivo consequence of bacterial cell death. Small molecules that induce similar perturbation in the enzyme-DNA complex should be candidates as leads for novel antibacterial agents. Second site mutations that can enhance the cell killing property of topoisomerase I are being studied. The SOS-inducing mutant topoisomerase I is useful as a model to study the mechanism of cell killing initiated by accumulation of topoisomerase I-mediated DNA cleavage.