The RecBCD enzyme from Escherichia coli is a complex multifunctional enzyme which performs critical functions in recombination and repair of the cellular DNA. The purified enzyme consists of three subunits encoded by the recB, recC, and recD genes and has ATP-dependent nuclease activity, DNA-dependent ATPase activity, and DNA helicase (unwinding) activity. The overall goal of this research is to understand at the molecular level the way in which the three enzyme subunits interact with each other and with the ATP and DNA substrates to catalyze these reactions. The research described in this proposal examines the specific functions of the individual subunits. The RecB, RecC, and RecD proteins will be purified and studied individually and in combination. Their properties will be investigated using a variety of assay methods for DNA binding, ATP hydrolysis, DNA unwinding, nuclease activity, and processivity. The results will be compared to those obtained previously with the RecBCD holoenzyme, the RecBC enzyme, and two mutant enzymes we have prepared, called RecB(K29Q) -CD and Rec3CD-K177Q. These mutants have a lysine-to- glutamine mutation in the putative ATP binding site in the RecB and RecD subunit, respectively. The single-stranded DNA-dependent hydrolysis of ATP and other nucleoside triphosphates catalyzed by the RecBCD and RecB(K29Q)CD enzymes will also be studied. A double mutant enzyme, with the lysine-to-glutamine change in both RecB and RecD, will also be prepared. The subunits of the enzyme bound directly to the DNA will be investigated by photo-cross-linking using DNA substituted with 5-bromo- deoxyuridine in place of thymidine. Ultraviolet irradiation from a laser light source will induce cross-linkages between the DNA and protein. The reaction conditions will be those in which the enzyme binds tightly to the DNA and acts on it processively, and where the DNA binds less tightly to the ends of the DNA and is unable to act as a helicase. These experiments will test hypotheses in which the enzyme binds the DNA at more than one site during the reactions. Cross-linking to DNA molecules containing cross-linking groups in only one strand or at specific positions in one strand will provide more detailed information about the architecture of the enzyme-DNA complexes.
