Site-specific DNA recombination is the process by which defined segments of DNA are broken and then rejoined to form different linkages at well defined sites. The Int-family of site-specific recombinases is a group of recombinases that share similar structures and mechanisms. These systems are used for a large number of different biological roles including control of gene expression (Hin), induction of DNA replication (FLP), reduction of dimeric DNA (Xer), and integration/excision of accessory elements (Int). The biological role of the Cre recombinase is to monomerize bacteriophage P1 DNA, although the system has become an important tool for the genomic manipulation of higher eukaryotes. Recombination site polarity is a common feature that is critical for the biological function and practical application of these systems. Specifically, the DNA recombination sites have a defined polarity and the recombination machinery is able to recognize that polarity to produce recombinant products with well defined structures. The goal of this proposal is to understand what features of the DNA substrates determine polarity and how the recombinase uses these features to control directionality? The proposal emphasizes the use of chemically modified DNA substrates to trap intermediates and isolate individual kinetic steps in the reaction. The Cre/lox system is particularly useful for addressing these questions because the DNA substrates (lox sites) can be chemically synthesized, the protein (Cre) is easily produced and purified, the reaction requires no accessory factors, and several crystal structures are available. The project will develop new tools that can be applies to many other systems. The result will test current models of recombination and will have important implications for the development of additional Cre-catalyzed reactions in vitro and in vivo.
