Structural Studies of RecA-DNA Complexes

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DESCRIPTION (provided by applicant): The overall goal of the proposed research is to use x-ray crystallography and other biochemical tools to understand the structure/function relationships of the E. coil RecA protein, the primary player in homologous recombination (HR). HR is a highly conserved, fundamental biological process that involves Ithe exchange of single-stranded DNA with one strand of a homologous region of duplex DNA. This process is becoming increasingly recognized as an important pathway for the repair of double-stranded DNA breaks, which are frequently generated during DNA replication and by environmental factors. HR is also potentially useful as a tool in the treatment of genetic disorders by gene therapy. RecA, a DNA-dependent ATPase, catalyzes the central strand-exchange step of HR by forming a helical polymer on ssDNA, facilitating a search for a homologous region of duplex DNA, and exchanging strands. E. coil RecA is 30% identical in amino acid sequence to the human enzyme (Rad51) and therefore will have a closely related biochemical mechanism. While the genetics and biochemistry of RecA have been studied for years, there is a major gap in the structural biology of RecA, and many aspects of the biochemical mechanism remain poorly understood. Although there is a x-ray structure of RecA in a helical form, the structure does not include DNA, and therefore does not show how the protein interacts with ssDNA and dsDNA substrates. This proposal outlines several approaches towards crystallizing RecA in complex with its ssDNA substrate. The work is also aimed at crystallizing RecA in the various conformational states, depending on the bound nucleotide, that are relevant to its catalytic mechanism. This work is important not only because the closely related human enzymes are relevant to disease, but also because the biochemical activity of the RecA protein is of fundamental importance to a broad class of enzymes- the helicases- that use a RecA-like structural scaffold to couple the energy of ATP-hydrolysis to DNA unwinding. Thus, understanding the mechanism of action of RecA will contribute greatly to our general knowledge of enzymes involved in virtually all aspects of DNA metabolism.