The broad, long-term objective of the proposed research to understand the molecular mechanism of general recombination. The biological systems to be examined are those of bacteriophage P22 and its relative, bacteriophage lambda. In these prokaryotic systems, general recombination plays an important role in protection of the chromosome from damage. It is likely that the basic principles of recombination/repair are the same in phages as in experimentally less accessible higher eukaryotes. Understanding these principles, in turn, is important in understanding human diseases, such as cancer, in which the underlying cause is DNA damage. A biochemically-oriented, in vitro approach is proposed, based upon the findings of earlier genetic, in vivo studies. Experiments will focus on the activities of these systems themselves, and on their interactions with the host cells recombination system, particularly RecBCD.
Specific aims i nclude; Determination of optimum conditions for lambda Red-mediated recombination in an in vitro system; establishment of system requirements by omission experiments; similar experiments with the P22 recombination system. Attempting to stage the in vitro reaction pathway through the isolation of complexes or intermediates formed in earlier stages, followed by addition of components needed at later stages; structural characterization of intermediates and products of recombination in vitro. Purification of genetically identified essential protein components of the system (if any are found in addition to the already-known RecA and lambda exonuclease + Redbeta (or P22's Abc-RecBCD + Erf + Arf). Fractionation of E. coli extracts for the purification of any unidentified essential components. Purification and characterization of Arf protein, especially with regard to effects on the DNA-binding activities of Erf and RecA. Attempts to obtain crystals of Erf and Redbeta proteins suitable for crystallographic studies; structure-function studies of Erf protein, in particular to learn about the function of the carboxy-terminal domain. Purification and characterization of Abc1-RecBCD and Abc1-Abc2-RecBCD; comparison of their biochemical activities with those of RecBCD, Abc2- RecBCD, and Gam-RecBCD; studies of the biochemistry of Abc2-RecBCD, especially wit regard to the types of structures formed at double-stranded ends in the presence and absence of purified ERF protein, and participation with Erf in strand exchange reactions. Use of the in vivo lambda RFLP assay to test predictions for the requirements of the P22 recombination system based on the biochemical studies of Abc2-modified RecBCD. Characterization of the Gam-modified host factors) responsible for stimulation of recombination in the lambda RFLP recombination assay.
