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 nuclease activity is inhibited, but DNA unwinding continues, when the enzyme encounters a particular DNA sequence called Chi (5'-GCTGGTGG). The overall goal of this research is to understand at the molecular level the way that the three enzyme subunits interact with each other, and with the ATP and DNA substrates, to catalyze these reactions. The biologically important functions of the RecBCD enzyme require all three of the proteins for maximal rate and efficiency. Thus, although both the RecBCD and RecD subunits have some DNA helicase activity, and the RecB protein alone has a low level of nuclease activity, all three subunits are required for the very rapid ATP-dependent nuclease and helicase activities characteristic of the RecBCD enzyme. The proposed research takes a combined genetics and enzymological approach. A 30 kD C-terminal domain of the RecB protein is required for the nuclease activity of RecBCD, but not for DNA unwinding, and it may contain the nuclease active site. Random but targeted mutations will be generated in this region of the RecB protein, and in the RecD protein, and mutants that specifically lack nuclease activity will be selected. The mutant proteins will then be purified and examined for their enzymatic properties by themselves and when complexed with the other subunits. The interactions among the protein subunits will be investigated using the yeast two-hybrid system, an in vivo genetics method. Mutations will be isolated that disrupt the interactions, and their effects on the enzymatic activities will be examined. Finally, the functions of the two ATPease subunits, RecB and RecD, in DNA unwinding by the enzyme will be investigated by studying the steady-state and pre-steady-state kinetics of nucleotide hydrolysis and DNA unwinding. These experiments will be done using the wild-type RecBCD enzyme, the RecBC enzyme, and a mutant enzyme that we prepared previously, RecBCD-K177Q, which has a mutation in the ATP binding site of the RecD subunit.
