The mechanism of generalized genetic recombination carried out by the bacteriophage P22 essential recombination function (erf gene) will be studied by biochemical and genetic means. The interaction of purified Erf protein with DNA will be examined by gel electrophoretic techniques and enzymatic probes of the state of the DNA. Fragments of the Erf protein, generated by proteolysis in vitro and by chain terminating mutations in vivo, will be purified. Their structural and DNA binding activities will be examined in parallel with those of intact Erf. The abc gene, a newly discovered P22 recombination-related gene, will be sequenced, and expressed at high levels by genetic engineering techniques. The Abc protein will be purified and its interaction with DNA, Erf protein, and, possibly, host cell recombination functions will be examined. Similar studies of Y, another new recombination-related gene of P22, will be carried out. A direct physical assay of erf-dependent homologous recombination will be developed and used to determine which phage and host cell components are necessary for this process. Attempts will be made to assay this process in vitro, in order to permit reconstruction of the erf recombination system from purified components. Such a reconstruction would greatly facilitate studies of mechanism. The long-term goal of this research is an understanding of the mechanisms of genetic recombination. Recombination is a process of fundamental importance in the generation of genetic variation and, in higher organisms, of diversity in the immune response.
