Bacteriophage Integrase (Int) is the prototype of a large family of tyrosine recombinases that perform recombination between specific pairs of target sequences. This family includes bacterial, phage, conjugative transposon, integron and yeast plasmid enzymes, and is structurally homologous and mechanistically similar to the eukaryotic type Ib topoisomerases, including those of Vaccinia virus and humans. The bacteriophage enzymes mediate regulated and directional recombination in response to the lysogeny/lysis decision of the phage to integrate into or excise from the host's genome. Thus, a hallmark of the phage enzymes is the ability to mediate the same catalytic events in the context of distinct higher-order complexes to yield different outcomes. lambda Int protein performs recombination through 4 distinct recombination pathways. Specific higher order complexes in each pathway somehow dictate whether the recombination reactions are unidirectional (in which products differ from substrates and the products cannot recombine to reform substrates) or bidirectional (in which the products have the same structure as the substrates). The long term goal of this project is to understand the molecular basis of directionality by determining the rate limiting step and comparing the geometry of central reaction intermediates in each pathway. A comparison will be made of one of the unidirectional reactions, excisive recombination, with an efficient bidirectional reaction, bent-L recombination. The investigator has isolated inhibitory peptides that block recombination before DNA cleavage or stabilize Holliday junctions. These will be used to modified DNA substrates to trap the cleaved but unligated Int-DNA covalent complex. These tools will be combined with atomic force microscopy to study the conformation of large nucleoprotein complexes containing pairs of DNA molecules, several Int monomers, and up to 4 accessory proteins. It is hoped that the work will lead to a detailed understanding of the recombination mechanisms as well as provide insights into the mechanisms of topoisomerases in general. The insights gained will be applicable to the development of antibiotics and cancer therapies and to the design of a higher diversity of improved gene targeting systems.
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