The general aim of the proposed project is to understand the mechanism by which the specificity of the site-specific recombination systems changes during evolution. The Cre protein and its recombinational target, the lox sites of bacteriophage P1 will serve as an experimental model. We will build a multipurpose experimental system by which we plan to identify specific nucleotides within the loxsites and specific amino acids in the Cre protein, responsible for specificity. To achieve this goal we will carry out systematic mutagenesis of the regions suspected to contribute to specificity, both in the sites and in the recombinase and will measure their recombinational properties. Based on this information we intend to change the specificity of the Cre/lox system by combining mutually compatible mutations in the lox sites and in the protein. If successful, this will answer the question how well we understood the evolutionary mechanisms. As a practical outcome, it will provide new varieties of the Cre/lox system with different specificities, increasing its utility to manipulate eukaryotic cells for research purposes.
