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J. Biol. Chem., Vol. 282, Issue 43, 31228-31237, October 26, 2007

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The Bacteroides NBU1 Integrase Performs a Homology-independent Strand Exchange to Form a Holliday Junction Intermediate^*

Lara Rajeev¹, Anca Segall, and Jeffrey Gardner

From the Department of Microbiology, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 and the Biology Department and Center for Microbial Sciences, San Diego State University, San Diego, California 92182

The Bacteroides mobilizable transposon NBU1 uses an integrase (IntN1) that is a tyrosine recombinase for its integration and excision from the host chromosome. Previously we showed that IntN1 makes 7-bp staggered cuts within the NBU1 att sites, and certain mismatches within the crossover region of the attN1 site (G(–2)C attN1) or the chromosomal target site (C(–3)G attBT1-1) enhanced the in vivo integration efficiency. Here we describe an in vitro integration system for NBU1. We used nicked substrates and a Holliday junction trapping peptide to show that NBU1 integration proceeds via formation of a Holliday junction intermediate that is formed by exchange of bottom strands. Some mismatches next to the first strand exchange site (in reactions with C(–3)G attBT1-1 or G(–2)C attN1 with their wild-type partner site) not only allowed formation of the Holliday junction intermediate but also increased the rate of recombinant formation. The second strand exchange appears to be homology-dependent. IntN1 is the only tyrosine recombinase known to catalyze a reaction that is more efficient in the presence of mismatches and where the first strand exchange is homology-independent. The possible mechanisms by which the mismatches stimulate recombination are discussed.

Received for publication, June 29, 2007 , and in revised form, August 27, 2007.

^* This work was supported by the National Institutes of Health NIGMS 28717 and 52847 and NIAID 58253. 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: Department of Microbiology, University of Illinois at Urbana-Champaign, 601 S. Goodwin Ave, B322 CLSL, Urbana, IL 61801. Tel.: 217-333-7289; Fax: 217-244-6697; E-mail: rajeev{at}uiuc.edu.

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