Tuberculosis is the leading cause of death from a single infection agent, i.e. Mycobacterium tuberculosis. The analysis of mycobacterial genetics and the development of strategies for improved diagnosis prevention and cure of mycobacterial diseases, will be facilitated by analysis of mycobacteriophages, viruses of the mycobacteria. The best-characterized of these viruses is mycobacteriophage L5, a temperate phage that forms stable lysogens in Mycobacterium smegmatis. Establishment of L5 lysogeny is accompanied by integration of the L5 genome into the mycobacterial chromosome. This event is catalyzed by the phage-encoded integrase protein which mediates site-specific recombination between the phage attachment, attP, and the chromosomal attachment site, attB. This recombination reaction has been established in a simple in vitro reaction and shown to require only attP DNA, attB DNA, integrase and a host factor protein encoded by M. smegmatis. This simple reaction involve several processes that are found in many other macromolecular processes in the cell. The integrase protein must recognize and bind to specific DNA sequences, and, with the assistance of the host factor, assemble a specific protein-DNA complex in which the two DNA partners come together in close proximity. An efficient and precise attack on the DNA must then occur followed by physical exchange of one set of DNA strands, rearrangement, and exchange of the second set of strands. To understand how these processes work, the L5 integrase protein and its interaction with the DNA sites will be examined. The nature of specific Protein-DNA complexes will be determined through analysis of the protein-DNA and protein-protein interactions, and the DNA site requirements. The action of the integrase Protein will be analyzed through the isolation of defective variants of the protein and characterizing them in vitro. The properties of the attP and attB attachment sites that confer their identity win also be investigated. In addition to Providing new insights into the mechanism of recombination these studies will offer new tools for mycobacterial genetics and a greater understanding of mycobacterial physiology.
|Lewis, John A; Hatfull, Graham F (2003) Control of directionality in L5 integrase-mediated site-specific recombination. J Mol Biol 326:805-21|
|Ghosh, Pallavi; Kim, Amy I; Hatfull, Graham F (2003) The orientation of mycobacteriophage Bxb1 integration is solely dependent on the central dinucleotide of attP and attB. Mol Cell 12:1101-11|
|Lewis, J A; Hatfull, G F (2001) Control of directionality in integrase-mediated recombination: examination of recombination directionality factors (RDFs) including Xis and Cox proteins. Nucleic Acids Res 29:2205-16|