The recBCD enzyme from E. coli functions in homologous recombination and repari of the cellular DNA. These processes are of fundamental importance in all organisms including man, where they contribute to the generation of antibody diversity and the activation of some proto-oncogenes leading to cancer. The enzyme consists of three subunits and has ATP-dependent nuclease activity on single- and double-stranded DNA, DNA-dependent ATPase activity, and DNA helicase (unwinding) activity. The overall goals of this project are to understand the mechanism by which the three subunits of this protein machin carry out this complex series of reactions on the DNA substrate, and how the enzyme participates in homologous recombination.
The specific aims of this proposal are 1) to determine the role in enzymatic acitivity of ATP-binding sites identified previously in the recB and recD subunits, and 2) to investigate the activity of the recBCD enzyme in the presence of the single-stranded DNA binding (SSB) and recA proteins from E. coli, and the coupled activities of the three proteins as an in vitro model system for recombination. The function of the ATP binding sites will be studied by examining the processivity of the nuclease reaction as a function of ATP, UTP, and non-hydrolyzable ATP analogues. The ATPase and UTPase reaction kinetics will also be studied as a function of DNA size and concentration. Amino acids believed to be directly involved in ATP binding by either the recB or recD subunit will also be altered by site-directed mutagenesis techniques. The activities and kinetic properties of the mutant enzymes will then be compared to those of the wild-type enzyme. The activities of the recBCD enzyme will also be investigated in the presence of the SSB and/or recA proteins. The mechanism by which SSB inhibits the nuclease activity will be elucidated by examining the kinetics, products, and specificity of reactions containing recBCD and SSB. Similar experiments will also be done with the recA prot alone in the presence of SSB. Finally, the three proteins will be tested for thir ability to promote reactions in vitro similar to those believed to occur in recombination in vivo. Linear and circular double-stranded DNA molecules sharing some sequences will be treated with recBCD enzyme, SSB and recA. The recA protein should be able to promote base-pairing between single-stranded DNA produced by recBCD nuclease or unwinding activity and homologous sequences in the circular molecule.
