title: 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.
authors.affiliation: Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA.
authors.forename: Bokun
authors.initials: B
authors.lastname: Cheng
authors.forename: Shikha
authors.initials: S
authors.lastname: Shukla
authors.forename: Sarinnapha
authors.initials: S
authors.lastname: Vasunilashorn
authors.forename: Somshuvra
authors.initials: S
authors.lastname: Mukhopadhyay
authors.forename: Yuk-Ching
authors.initials: YC
authors.lastname: Tse-Dinh
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000890:Anti-Infective Agents / Q000494:pharmacology*; D001089:Arabinose / Q000737:chemistry; D001665:Binding Sites; D002384:Catalysis; D004247:DNA / Q000737:chemistry; D004264:DNA Topoisomerases, Type I / Q000737:chemistry; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005047:Etoposide / Q000494:pharmacology; D008274:Magnesium / Q000494:pharmacology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016296:Mutagenesis; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010957:Plasmids / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D014364:Tryptophan / Q000737:chemistry; D015010:Yersinia pestis / Q000201:enzymology
chemical_list: D000890:Anti-Infective Agents; D011994:Recombinant Proteins; D005047:Etoposide; D014364:Tryptophan; D004247:DNA; D001089:Arabinose; D004264:DNA Topoisomerases, Type I; D008274:Magnesium