Bacterial Cell Killing Mediated by Topoisomerase I DNA Cleavage Activity

Abstract

DNA topoisomerases are important clinical targets for antibacterial and anticancer therapy. At least one type IA DNA topoisomerase 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. However, it has not been possible previously to observe the consequence of having such a stabilized covalent complex of bacterial topoisomerase I in vivo. We isolated a mutant of recombinant Yersinia pestis topoisomerase I that forms a stabilized covalent complex with DNA by screening for the ability to induce the SOS response in Escherichia coli. Overexpression of this mutant topoisomerase I resulted 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 E. 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.DNA topoisomerases are ubiquitous enzymes that are needed either for control of DNA supercoiling or for overcoming topological barriers during replication, transcription, recombination, or repair (reviewed in Refs. 1-3). Type IB and type II DNA topoisomerases are well utilized targets of many clinically important anticancer and antibacterial drugs (4 -7). These drugs cause cell death by stabilizing the covalent intermediate formed between topoisomerase protein and cleaved DNA during the catalytic cycle of the enzyme. There is at least one type IA DNA topoisomerase present in every genome examined so far. It is likely to be essential for overcoming topological barriers requiring single-stranded DNA passage (2). It has been proposed that bacterial type IA DNA topoisomerases could be a useful therapeutic target if small molecules that stabilize the covalent intermediate of this class of topoisomerases can be identified (8). However, it has never been demonstrated that stabilization of the covalent intermediate formed between a type IA topoisomerase and the cleaved single DNA strand can lead to cell death. It therefore remains unclear whether bacterial topoisomerase I can indeed be the target of "poison" molecules that would be bactericidal.In this study, a mutant bacterial topoisomerase I that forms a stabilized covalent intermediate with cleaved DNA was identified via an SOS induction screen (9) in Escherichia coli. Overexpression of this mutant topoisomerase in E. coli led to extensive cell ki...

The Strictly Conserved Arg-321 Residue in the Active Site of Escherichia coli Topoisomerase I Plays a Critical Role in DNA Rejoining

“…The Y. pestis topoisomerase I protein sequence shares 85% identity with E. coli topoisomerase I protein sequence, so mutations at analogous positions have been shown to have similar effects in both enzymes (22,28,32). Mutations affecting DNA rejoining are expected to be highly lethal due to accumulation of the cleaved covalent complex, and recombinant clones of such Y. pestis topoisomerase I mutants were found to be more stably maintained in E. coli than analogous recombinant E. coli topoisomerase I mutant clones.…”

“…Identification of Y. pestis Topoisomerase I Mutants with Deficiency in DNA Rejoining-E. coli strain JD5 containing the chromosomal dinD1::lacZ fusion as SOS response reporter was transformed with plasmid pYTOP expressing Y. pestis topoisomerase I (YpTOP1) 2 under the control of the BAD promoter (22). Random mutations were introduced into the YpTOP1 coding sequence by PCR (22).…”

“…Random mutations were introduced into the YpTOP1 coding sequence by PCR (22). The transformants were first isolated on Luria-Bertani (LB) plates with 100 g/ml ampicillin and 2% glucose to suppress the expression of potentially lethal recombinant mutant YpTOP1, and then replica plated onto X-Gal indicator plates containing 0.002% arabinose and 100 g/ml ampicillin to induce the expression of the recombinant topoisomerase I (22). Mutant clones that induce the SOS response were identified as blue colonies and maintained in LB with 2% glucose and 100 g/ml ampicillin.…”

“…These potentially dominant lethal mutations in recombinant Y. pestis topoisomerase I can be more stably maintained in E. coli than the corresponding E. coli topoisomerase I mutants. Mutation of the glycine in the TOPRIM motif to serine was first identified for inhibition of DNA religation and enhanced cell killing due to accumulation of the covalent cleavage complex (22). The substitution of the first aspartate residue in the TOPRIM motif with asparagine has been shown to be an even more lethal mutation (23), demonstrating the requirement of Mg 2ϩ for DNA religation even though Mg 2ϩ is not absolutely required for DNA cleavage.…”

“…Induces SOS Response-The coding sequence of Y. pestis topoisomerase I gene under the control of the BAD promoter in plasmid pYTOP was subjected to random mutagenesis (22). The mutated pYTOP DNA was then transformed into E. coli strain JD5, which has a chromosomal dinD1::lacZ fusion that produces ␤-galactosidase when the SOS response is activated due to DNA damage (30).…”