The goal of the work in this proposal is to understand how DNA recombinase enzymes function at the molecular level. Our studies will focus on the bacterial RecA and human Rad5l (HsRad5l) proteins whose activities are of fundamental importance for the maintenance of genomic integrity. In bacteria, RecA is the central regulatory and catalytic component of a DNA-damage inducible recombinational repair system. In humans and other vertebrates, Rad5l is essential for survival and is a central component of a complex set of proteins involved in the catalysis of homologous genetic recombination and recombinational DNA repair. We have recently characterized an unexpected difference between RecA and HsRad5l regarding the regulation of their activity by ATP-mediated allosteric events and will exploit this finding in developing further studies of HsRad5l. We will use a combination of genetic and biochemical methods to address specific mechanistic questions about each enzyme. Studies are also designed to understand how the interaction of HsRad5l with other recombination proteins contributes to its function. Our work addresses questions regarding the molecular nature of conserved aspects of bacterial and human homologous recombination, while at the same time identifying features unique to the human proteins. Understanding the underlying molecular mechanistic principles of such proteins has far-reaching effects in ultimately being able to create novel proteins with desired properties that may be used for prevention of diseases resulting from genomic instability and for beneficial genetic manipulation.
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