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.

National Institute of Health (NIH)
National Institute of Allergy and Infectious Diseases (NIAID)
Research Project (R01)
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Microbial Physiology and Genetics Subcommittee 2 (MBC)
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University of Massachusetts Medical School Worcester
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Murphy, K C (1998) Use of bacteriophage lambda recombination functions to promote gene replacement in Escherichia coli. J Bacteriol 180:2063-71
Murphy, K C (1994) Biochemical characterization of P22 phage-modified Escherichia coli RecBCD enzyme. J Biol Chem 269:22507-16
Ranade, K; Poteete, A R (1993) A switch in translation mediated by an antisense RNA. Genes Dev 7:1498-507
Ranade, K; Poteete, A R (1993) Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda. J Bacteriol 175:4712-8
Poteete, A R; Fenton, A C (1993) Efficient double-strand break-stimulated recombination promoted by the general recombination systems of phages lambda and P22. Genetics 134:1013-21
Murphy, K C; Lewis, L J (1993) Properties of Escherichia coli expressing bacteriophage P22 Abc (anti-RecBCD) proteins, including inhibition of Chi activity. J Bacteriol 175:1756-66
Poteete, A R; Fenton, A C; Semerjian, A V (1991) Bacteriophage P22 accessory recombination function. Virology 182:316-23
Murphy, K C (1991) Lambda Gam protein inhibits the helicase and chi-stimulated recombination activities of Escherichia coli RecBCD enzyme. J Bacteriol 173:5808-21
Hardy, L W; Poteete, A R (1991) Reexamination of the role of Asp20 in catalysis by bacteriophage T4 lysozyme. Biochemistry 30:9457-63
Semerjian, A V; Malloy, D C; Poteete, A R (1989) Genetic structure of the bacteriophage P22 PL operon. J Mol Biol 207:1-13

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