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J. Biol. Chem., Vol. 280, Issue 46, 38489-38495, November 18, 2005

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Bacterial Cell Killing Mediated by Topoisomerase I DNA Cleavage Activity^*

Bokun Cheng, Shikha Shukla, Sarinnapha Vasunilashorn, Somshuvra Mukhopadhyay, and Yuk-Ching Tse-Dinh1

From the Department of Biochemistry and Molecular Biology and the Department of Cell Biology and Anatomy, New York Medical College, Valhalla, New York 10595

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.

Received for publication, September 2, 2005 , and in revised form, September 8, 2005.

^* This work was supported by National Institutes of Health Grants R01 GM54226 and R03 NS050782 and Grant C-020219 from the New York State Department of Health administered by the Northeast Biodefense Center (to Y.-C. T.-D.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, New York Medical College, Valhalla, NY 10595. Tel.: 914-594-4061; Fax: 914-594-4058; E-mail: yuk-ching_tse-dinh{at}nymc.edu.

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				E. P. Sorokin, B. Cheng, S. Rathi, S. J. Aedo, M. V. Abrenica, and Y.-C. Tse-Dinh Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region Nucleic Acids Res., August 1, 2008; 36(14): 4788 - 4796. [Abstract] [Full Text] [PDF]

				J. H. Sutherland, B. Cheng, I-F. Liu, and Y.-C. Tse-Dinh SOS Induction by Stabilized Topoisomerase IA Cleavage Complex Occurs via the RecBCD Pathway J. Bacteriol., May 1, 2008; 190(9): 3399 - 3403. [Abstract] [Full Text] [PDF]

				B. Cheng, E. P. Sorokin, and Y.-C. Tse-Dinh Mutation adjacent to the active site tyrosine can enhance DNA cleavage and cell killing by the TOPRIM Gly to Ser mutant of bacterial topoisomerase I Nucleic Acids Res., February 11, 2008; 36(3): 1017 - 1025. [Abstract] [Full Text] [PDF]

				B. Cheng, I-F. Liu, and Y.-C. Tse-Dinh Compounds with antibacterial activity that enhance DNA cleavage by bacterial DNA topoisomerase I J. Antimicrob. Chemother., April 1, 2007; 59(4): 640 - 645. [Abstract] [Full Text] [PDF]