Site-specific recombination is the reciprocal exchange of DNA catalyzed by specific recombination proteins acting at specific DNA sites. Study of such processes provides information on the nature of protein-DNA recognition and interaction, the nature of macromolecular complexes that act with great precision, and the mechanism by which specific enzymes cleave and exchange DNA strands. This information is relevant to many biological processes including other types of DNA recombination, DNA replication and transcription. This project will examine integration and excision of mycobacteriophage L5 DNA, events that play important roles in the life cycles of the virus. The integrative reaction involves a specific phage attachment site, attP and a specific chromosomal site, attB and is catalyzed by the L5-encoded protein, integrase. This integrative reaction provides an important tool for the construction of multidisease recombinant vaccines. To better understand the nature of L5 recombination, the reaction will be reconstituted in an in vitro system in which the requirements for DNA, protein and protein-DNA interactions can be evaluated. This initial characterization will delineate novel aspects of the reaction including the number and location of specific DNA binding sites, the activities and properties of the integrase protein and isolation and characterization of host proteins that may participate or stimulate recombination. Following initial characterization of the in vitro reaction, experiments will focus on understanding the integrase protein that catalyzes the event. Two approaches will be used. First, we will isolate and characterize mutant forms of integrase that are unable to mediate recombination, thus determining the roles of specific amino acid residues. Second, we will attempt to determine the macromolecular structure of integrase and integrase-DNA complexes using crystallographic methods. These experiments will provide new insights into the mechanism of site- specific recombination and the formation of macromolecular complexes.
|Parish, T; Lewis, J; Stoker, N G (2001) Use of the mycobacteriophage L5 excisionase in Mycobacterium tuberculosis to demonstrate gene essentiality. Tuberculosis (Edinb) 81:359-64|