The goal of the research is to further the development and analysis of a simple prokaryotic system which models the complex process of homologous recombination in eukaryotes. The investigators have replaced the normal recombination system of the bacterium Escherichia coli with the Red recombination system of bacteriophage lambda. This switch to the Red system makes the bacterium far more prone to carry out recombination events involving linear double-stranded DNA molecules in the size range of single genes. The recombining DNA will be delivered into the Red-expressing bacteria by infection with a modified version of bacteriophage lambda. This bacteriophage injects its chromosome, which is cut in two places by a restriction endonuclease present in the bacterium, releasing a linear double stranded DNA molecule containing the antibiotic resistance gene cat, flanked by sequences identical to parts of the lacZ gene in the bacterial chromosome. Recombination between the linear DNA and the circular bacterial chromosome produces a strain in which lacZ is replaced by cat. Unlike the starting strain, these recombinants are both antibiotic-resistant and unable to utilize the sugar lactose; they are readily selected and enumerated. Experiments are proposed to explore the involvement of various recombination-promoting genes in Red-mediated gene replacement. The bacterial genes recF, recO, recR, recA, and recQ, and the lambda genes orf and rap will be studied in particular. In a second set of experiments, the dependence of the recombination event upon structural features of the linear recombining DNA species will be examined. Questions to be addressed include whether the system responds more efficiently to some sequences than to others, and whether various artificial DNAs bearing indigestible phosphorothioate linkages are active in Red-mediated recombination.
