The RecBCD enzyme acts in homologous DNA recombination in Escherichia coil and other bacteria. Homologous recombination is essential for the repair of some types of DNA damage and is an important part of the DNA replication process. Bacteria with mutations in the recB or recC genes are deficient in their ability to carry out homologous recombination and they have low viability. That is, a large fraction of the cells are inviable after cell division as a result of the inability to repair chromosome breaks that arise during DNA replication. Homologous recombination is also important in DNA repair and replication in higher organisms including humans. Defects in these processes have significant negative health implications, as they can be a contributing factor in the development of diseases including cancer. RecBCD is also quite interesting as a multisubunit enzyme machine. It is a multifunctional enzyme that unwinds and degrades linear DNA as an ATP-dependent exonuclease. The enzyme activity is regulated by a specific DNA sequence that has been called Chi (5'-GCTGGTGG). An encounter with Chi affects the rate and strand specificity of the nuclease reaction. All of these phenomena are critically dependent on the reaction conditions, especially the ATP and magnesium ion concentrations. The principal investigator has studied the enzyme by dissecting it into its subunits and protein domains and characterized the activities of those component parts. This proposal describes enzymological experiments to study how the enzyme activities and structure are affected by ligands including magnesium ion, DNA, and ATP, to learn more about the regulation of the enzyme activity that happens at the Chi sequence. He will also study in more detail the structure of a small domain of the RecB subunit that has the nuclease active site, how its activity is affected by the presence of the other proteins, and comparative experiments with another nuclease that has a similar active site sequence.
