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J. Biol. Chem., Vol. 283, Issue 18, 12402-12414, May 2, 2008

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A Biotin Interference Assay Highlights Two Different Asymmetric Interaction Profiles for Integrase Arm-type Binding Sites in Integrative Versus Excisive Recombination^*

Dane Hazelbaker, Marco A. Azaro, and Arthur Landy¹

From the Department of Molecular Biology, Cellular Biology, and Biochemistry, Brown University, Providence, Rhode Island 02912 and the Department of Genetics, Rutgers University, Piscataway, New Jersey 08854

The site-specific recombinase integrase encoded by bacteriophage promotes integration and excision of the viral chromosome into and out of its Escherichia coli host chromosome through a Holliday junction recombination intermediate. This intermediate contains an integrase tetramer bound via its catalytic carboxyl-terminal domains to the four "core-type" sites of the Holliday junction DNA and via its amino-terminal domains to distal "arm-type" sites. The two classes of integrase binding sites are brought into close proximity by an ensemble of accessory proteins that bind and bend the intervening DNA. We have used a biotin interference assay that probes the requirement for major groove protein binding at specified DNA loci in conjunction with DNA protection, gel mobility shift, and genetic experiments to test several predictions of the models derived from the x-ray crystal structures of minimized and symmetrized surrogates of recombination intermediates lacking the accessory proteins and their cognate DNA targets. Our data do not support the predictions of "non-canonical" DNA targets for the N-domain of integrase, and they indicate that the complexes used for x-ray crystallography are more appropriate for modeling excisive rather than integrative recombination intermediates. We suggest that the difference in the asymmetric interaction profiles of the N-domains and arm-type sites in integrative versus excisive recombinogenic complexes reflects the regulation of recombination, whereas the asymmetry of these patterns within each reaction contributes to directionality.

Received for publication, January 22, 2008 , and in revised form, March 3, 2008.

^* This work was supported by National Institutes of Health Grants GM62723 and GM33928 (to A. L.). 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 1.

¹ To whom correspondence should be addressed: 185 Meeting St., Providence, RI 02912. Tel.: 401-863-2571; Fax: 401-863-9583; E-mail: Arthur_Landy{at}brown.edu.

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