Enzymatic Mechanisms of DNA Helicases

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DESCRIPTION (provided by applicant): DNA helicases provide the primary mechanism by which duplex DNA is converted to single-stranded DNA (ssDNA) for use as a template in DNA replication and repair or as a substrate in recombination. Indeed, these enzymes are essential for DNA replication, repair and to maintain genomic stability in all organisms. Consistent with this idea, defects in genes encoding DNA helicases in human cells have been linked to genomic instability leading to a variety of progeriod disorders and human cancers. Thus, a complete understanding of the mechanisms and roles played by these enzymes is essential to understanding the fundamentals of DNA transactions within the cell. During the last grant period we have made significant progress in understanding the interaction between a mismatch repair protein, MutL, and UvrD. This interaction results in a dramatic stimulation of UvrD-catalyzed unwinding. We have shown that MutL helps load UvrD, that MutL must have its ATP cofactor bound to stimulate UvrD and we now have preliminary results indicating that MutL acts as a processivity factor to increase the length of duplex DNA unwound by UvrD. When fully confirmed by the kinetic and biophysical experiments proposed in aim 1, this will be the first well-described example of a helicase processivity factor. We have also dissected the domain structure of DNA helicase I encoded by the traI gene on the F plasmid. We have shown this helicase to be active as a momomer, extraordinarily processive and very fast. We will use single molecule experiments to directly demonstrate the processivity of the protein as proposed in aim 2. We will also use biochemical, biophysical and structural experiments to define the mechanism by which this protein catalyzes processive unwinding. This represents a unique opportunity to understand how a monomeric protein can achieve remarkable processivity. Finally, we have recently shown that UvrD unwinds Holliday Junction substrates, presumably initiating at the junction. We hypothesize that UvrD acts to destroy unwanted recombination intermediates containing a mismatch in a pathway that involves MutS and MutL, and is essential to ensure genomic stability. This new reaction and biological role for UvrD will be investigated using kinetic and biochemical experiments in aim 3. PUBLIC HEALTH RELEVANCE: DNA helicases are essential for DNA replication, repair, recombination and to maintain genomic stability in all organisms. Interest in fully understanding this class of proteins has been heightened by the discovery that mutations in specific DNA helicases result in several human genetic diseases. It is likely that other diseases will be linked with defects in DNA helicases as more disease genes are identified placing added emphasis on the importance of understanding this class of proteins.