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. The system to be studied is the homologous recombination system of bacteriophage P22, which consists of the protein products of four phage genes: Erf, the key component, a strand-exchanging protein; Arf (accessory recombination function), which enhances Erf activity in vivo; and Abc1 and Abc2 (anti-RecBCD), which modulate the activity of the host cell RecBCD protein, diverting it into the phage recombination pathway. The mechanism of homologous recombination carried out by the P22 system will be studied by biochemical and genetic means. In vivo assays of recombination between phages will pe employed to examine the role of double-chain breaks in the Erf system, and to test the activity of Arf in promoting homologous recombination by other systems. Individual components of the recombination system will be purified and characterized biochemically: structural and functional alteration of RecBCD by Abc (ana by the Gam protein of phage lambda), modulation of Erf activity by Arf, and the function of the carboxy-terminal domain of Erf will be examined in particular. Attempts will be made to assemble, from purified components in vitro, systems and sub-systems that faithfully mimic the activity of the P22 homologous recombination in vivo. The actions of these systems on substrate DNA will be studied by gel electrophoretic and electron microscopic techniques, with the aim of establishing the individual steps of the process and characterizing the intermediates formed.
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