The RecA protein is a multifunctional, oligomeric enzyme which is the central component of the processes of recombinational repair and homologous genetic recombination in E. coli. The long term goal of the research is to understand the functional organization and structural dynamics of RecA in order to determine how the many activities catalyzed by this enzyme are coordinated in carrying out the overall process of recombination. Dr. Knight will begin this work by identifying regions of the protein that are involved in particular functions, e.g. DNA binding, ATP binding and hydrolysis and oligomeric contacts by performing protein covalent modification experiments. Also, in collaboration with Dr. Thomas Steitz, Dr. Knight will use the recently solved X-ray crystal structure of RecA to aid in defining important functional and structural areas of the protein. Once regions of the protein have been identified as critical for function and/or structure by cross-linking studies, he will use a variety of mutagenic techniques to alter specific amino acids in that region. A number of genetic selections and screens will be used to differentiate between those mutant proteins which maintain catalytic abilities and those with different recombination activities. These mutant proteins will be analyzed for their ability to undergo ligand-induced conformational changes. Dr. Knight has previously demonstrated that the RecA protein undergoes an ATP-induced conformational change, and subsequent electron microscopic studies suggest that this structural change is critical to RecA function. Further genetic analysis of the relationship between protein structure and function will be performed by using a number of the recombination deficient mutant proteins obtained in this study to select intragenic second site revertants. These revertants will be analyzed to define further the domain structure of RecA protein.
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