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Nucleic Acids Research Advance Access published online on July 24, 2008
Nucleic Acids Research, doi:10.1093/nar/gkn460
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© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Nucleic Acid Enzymes

Inhibition of Mg2+ binding and DNA religation by bacterial topoisomerase I via introduction of an additional positive charge into the active site region

Elena P. Sorokin, Bokun Cheng, Siddarth Rathi, Sandra J. Aedo, Maria V. Abrenica and Yuk-Ching Tse-Dinh*

Department of Biochemistry and Molecular Biology, New York Medical College, Valhalla, New York 10595, USA

*To whom correspondence should be addressed. Tel: +1 914 594 4061; Fax: +1 914 594 4058; Email: yuk-ching_tse-dinh{at}nymc.edu

Received May 5, 2008. Revised June 26, 2008. Accepted July 2, 2008.

Among bacterial topoisomerase I enzymes, a conserved methionine residue is found at the active site next to the nucleophilic tyrosine. Substitution of this methionine residue with arginine in recombinant Yersinia pestis topoisomerase I (YTOP) was the only substitution at this position found to induce the SOS response in Escherichia coli. Overexpression of the M326R mutant YTOP resulted in ~4 log loss of viability. Biochemical analysis of purified Y. pestis and E. coli mutant topoisomerase I showed that the Met to Arg substitution affected the DNA religation step of the catalytic cycle. The introduction of an additional positive charge into the active site region of the mutant E. coli topoisomerase I activity shifted the pH for optimal activity and decreased the Mg2+ binding affinity. This study demonstrated that a substitution outside the TOPRIM motif, which binds Mg2+directly, can nonetheless inhibit Mg2+ binding and DNA religation by the enzyme, increasing the accumulation of covalent cleavage complex, with bactericidal consequence. Small molecules that can inhibit Mg2+ dependent religation by bacterial topoisomerase I specifically could be developed into useful new antibacterial compounds. This approach would be similar to the inhibition of divalent ion dependent strand transfer by HIV integrase in antiviral therapy.

Present address: Siddarth Rathi, Boston University School of Medicine, Boston, MA, USA Maria V. Abrenica, Wellesley College, Wellesley, MA, USA

The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors


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