The long term goal of this proposal is to determine the enzymatic mechanisms of genetic recombination between duplex DNA molecules in Escherichia coli. The mechanism of two different recombination pathways, the RecF and RecE pathways will be studied. The basic approach that will be followed is to develop in vitro recombination systems and biochemical assays that measure genetic recombination in order to identify the biochemical intermediates and purify the proteins that are involved in genetic recombination. The mechanism of RecF pathway recombination events in vivo will be studied in detail. In particular, the effect of double-strand breaks and other types of DNA damage will be determined. RecF pathway genes will be cloned and used to overproduce and purify the proteins that they encode. The recF protein, which has already been purified, will be characterized in detail and additional oveproduction studies will concentrate on the recJ, recO, and recQ proteins. A possible role for a Holliday junction resolution enzyme in RecF pathway recombination will be investigated. In vitro systems that use crude extracts and/or partially purified proteins to catalyze RecF pathway recombination reactions will be developed and the intermediates and products that are formed in these reactions will be characterized in detail. Individual RecF pathway proteins that were not purified as part of the overproduction studies will be purified using a combination of in vitro complementation and reconstitution assays and characterized in detail. The RecE pathway recombination will be studied using the same types of methods as those described for RecF pathway reactions. Additional studies to be carried out include genetic studies to identify new genes that are required for the RecE pathway and biochemical analysis of exonuclease VIII to define the functional domain of exonuclease VIII that is required for both RecE pathway recombination and exonuclease activity. The ultimate goal of these studies will be to reconstitute RecF and RecE pathway recombination reactions with purified proteins and determine the enzymatic mechanisms of these reactions.
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