HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 281, Issue 49, 38052-38060, December 8, 2006

This Article Full Text Full Text (PDF) TopoMutants8cleav.pdf Supplemental Data All Versions of this Article: 281/49/38052    most recent M608858200v1 Submit a Letter to Editor Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Hwang, Y. Articles by Bushman, F. D. Search for Related Content PubMed PubMed Citation Articles by Hwang, Y. Articles by Bushman, F. D. Social Bookmarking                      What's this?

Regulation of Catalysis by the Smallpox Virus Topoisomerase^*

Young Hwang, Nana Minkah, Kay Perry, Gregory D. Van Duyne¹, and Frederic D. Bushman²

From the Departments of Microbiology and Biochemistry & Biophysics and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania 19104

The poxvirus type IB topoisomerases catalyze relaxation of supercoiled DNA by cleaving and rejoining DNA strands via a pathway involving a covalent phosphotyrosine intermediate. Recently we determined structures of the smallpox virus topoisomerase bound to DNA in covalent and non-covalent DNA complexes using x-ray crystallography. Here we analyzed the effects of twenty-two amino acid substitutions on the topoisomerase activity in vitro in assays of DNA relaxation, single cycle cleavage, and equilibrium cleavage-religation. Alanine substitutions at 14 positions impaired topoisomerase function, marking a channel of functionally important contacts along the protein-DNA interface. Unexpectedly, alanine substitutions at two positions (D168A and E124A) accelerated the forward rate of cleavage. These findings and further analysis indicate that Asp¹⁶⁸ is a key regulator of the active site that maintains an optimal balance among the DNA cleavage, religation, and product release steps. Finally, we report that high level expression of the D168A topoisomerase in Escherichia coli, but not other alanine-substituted enzymes, prevented cell growth. These findings help elucidate the amino acid side chains involved in DNA binding and catalysis and provide guidance for designing topoisomerase poisons for use as smallpox antivirals.

Received for publication, September 14, 2006 , and in revised form, October 10, 2006.

^* This work was supported by a grant from the Mid-Atlantic Regional Center of Excellence for Biodefense Research (to F. D. B.). 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.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Table S1.

¹ An Investigator of the Howard Hughes Medical Institute.

² To whom correspondence should be addressed: Dept. of Microbiology, University of Pennsylvania School of Medicine, 402C Johnson Pavilion, 3610 Hamilton Walk, Philadelphia, PA 19104-6076. Tel.: 215-573-8732; Fax: 215-573-4856; E-mail: bushman{at}mail.med.upenn.edu.

CiteULike    Complore    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this?

This article has been cited by other articles:

				L. Yakovleva, S. Chen, S. M. Hecht, and S. Shuman Chemical and Traditional Mutagenesis of Vaccinia DNA Topoisomerase Provides Insights to Cleavage Site Recognition and Transesterification Chemistry J. Biol. Chem., June 6, 2008; 283(23): 16093 - 16103. [Abstract] [Full Text] [PDF]