SITE-SPECIFIC RECOMBINATION

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Site-specific recombination systems are important in controlling development and gene expression in a diverse array of organisms ranging from bacteria to humans. The long range goal of this project is to understand how bacteriophage lambda carries-out site-specific recombination. Both biochemical and genetic approaches will be used to characterize the protein-protein and protein-DNA interactions that occur during the assembly of recombination complexes (intasomes) and the process of strand exchange. The phage-encoded integrase (Int) protein participates in intasome formation and catalyzes strand exchange. Mutants with altered DNA binding specificities will be isolated to determine which amino acid residues in Int are responsible for DNA recognition. Other Int mutants will be isolated and characterized in biochemical assays that will determine the defects of individual mutant proteins in the recombination pathway. The host-encoded integration host factor (IHF) participates in intasome formation by inducing bends in the DNA. A combination of in vivo and in vitro mutagenesis approaches will be used to isolate and to characterize altered DNA binding specificity mutants of IHF. Such mutants will identify the amino acid residues in IHF that interact with DNA and will be useful in interpreting structures derived from physical studies. The phage-encoded excisionase (Xis) and the host-encoded factor for inversion stimulation (FIS) promoter excisive recombination. Xis interacts cooperatively with Int and FIS. Xis mutants will be isolated that are defective in cooperative interactions with Int and FIS and the mutant proteins will be used to determine the mechanism(s) of the cooperative interactions.