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J. Biol. Chem., Vol. 283, Issue 17, 11414-11423, April 25, 2008

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Binding and Catalytic Contributions to Site Recognition by Flp Recombinase^*

From the Department of Biochemistry and Molecular Biology, The University of Chicago, Chicago, Illinois 60637

Flp catalyzes site-specific recombination in a highly sequence-specific manner despite making few direct contacts to the bases within its binding site. Sequence discrimination could take place in the binding and/or the catalytic steps. In this study, we independently measure the binding affinity and initial cleavage rate of Flp recombinase with 20 designed alternate target DNA sequences. Our results show that Flp specificity is largely, although not entirely, imparted at the binding step and is the result of a combination of direct and indirect readout. The Flp binding site includes an A/T-rich region that displays a characteristically narrow minor groove. We find that many A T changes are tolerated at the binding step, whereas C or G substitutions tend to decrease binding affinity. The effects of the latter can be alleviated by replacing guanine with inosine, which removes the N2 amino group that protrudes into the minor groove. Some A T changes reduce binding affinity, due to clashing with nearby residues, reinforcing that specificity requires avoiding negative contacts as well as creating positive ones. A tracts, which can lead to unusually rigid DNA structure, are tolerated during the binding step when placed within the region where the minor groove is already narrow. However, most A tracts slow catalysis more than C or G substitutions. Understanding what kind of sequence variation is tolerated in the binding and catalytic steps helps us understand how the target DNA is recognized by Flp and will be useful in guiding the design of Flp variants with altered specificities.

Received for publication, January 4, 2008 , and in revised form, February 13, 2008.

^* This work was supported by National Institutes of Health Grant GM058827. 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: 929 E. 57th St., GCIS W136, Chicago, IL 60637. E-mail: price{at}uchicago.edu.

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