HOMOLOGOUS RECOMBINATION SYSTEMS OF PHAGES P1 & LAMBDA

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Long-term objectives are to understand how homologous recombination is enhanced in two situations where known recombination enzymes are in excess, or nearly so: RecA-mediated recombination in E. coli and recombination catalyzed by the phage Lambda "Red" system. Since uncontrolled genetic rearrangement may play a role in cancer and some genetic diseases, whereas "controlled" homologous recombination is an important source of biological diversity, it is important to understand the factors that affect recombination frequencies. Specific aims of the study of a novel function of phage PL ("ref") that stimulates certain RecABC-dependent homologous recombinations in E. coli are: (i) identification, purification, and characterization of the ref gene product; (ii) determination of the mechanism by which ref function promotes recombination; (iii) the general features of regulation of ref; (iv) the role of ref in Pl growth and lysogeny. Corresponding methodologies include: (i) cloning ref for overproduction, radiochemical purification, testing for likely activities, (ii) testing a variety of recombination processes for ref effects, mobilizing the known lacDelta substrates for ref onto Lambda phages; (iii) identification of putative negative and positive control elements by testing cloned Pl fragments in various regulatory situations, identification of P1 functions coregulated with ref; (iv) testing for phage growth, establishment and/or maintenance of lysogeny when E. coli homologous recombination and/or Pl site-specific recombination (cre/lox) is deficient. Specific aims of the Lambda Red studies are: (i) determination of which Lambda PL functions besides Lambda exonuclease and Beta protein (if any) are needed, in addition to Lambda replication functions, for recombination; (ii) development of an in vitro system; (iii) determination of the function of Lambda ssb protein; (iv) characterization of the Lambdagam-RecBC nuclease interaction in vivo. Corresponding methodologies include: (i) use of our exo bet lacPO plasmids, in conjunction with appropriate Lambdabio substitution phages for in vivo recombination studies with genetically marked phages lacking (nearly) all PL functions, and with Lambdadv plasmids; (ii) assay of in vitro Lamdadv recombination by electrophoretic, or more sensitive, methods; (iii) testing the effect of Lambdassb expression in E. coli ssb (ts) mutants; (iv) determining the effect of gam-overproducing plasmids on E. coli (rec+) UV-sensitivity and recombination proficiency.