Helicase Catalyzed DNA Unwinding

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DESCRIPTION (provided by applicant): DNA helicases are ATP-dependent motor proteins that unwind duplex DNA to form the single stranded (ss)DNA intermediates required for DNA metabolism in all organisms. We are studying the kinetic mechanisms of DNA unwinding and DNA translocation of three non-hexameric SF1 DNA helicases from E. coil, Rep,UvrD, and RecBCD, which function in replication, repair, and recombination, respectively. RecBCD is a hetero-trimeric complex containing two SF1 helicases (B and D). The overall goal is to obtain a molecular understanding of the kinetic mechanism(s) by which these DNA helicases unwind duplex DNA, translocate along DNA and how these processes are coupled to ATP binding and hydrolysis. Our pre-steady state kinetic studies indicate that Rep and UvrD helicases function as dimers, even though monomers of these enzymes can translocate efficiently along ss-DNA, hence the relationship between ss-DNA translocation by monomers and DNA unwinding by dimers will be investigated. We will use transient kinetic approaches(stopped-flow fluorescence and chemical quenched-flow) to examine the pre-steady state kinetics and mechanism of ss-DNA translocation, ATP binding and hydrolysis by Rep and UvrD monomers during ss-DNA translocation, and compare these to the kinetic mechanism for DNA unwinding by the functional helicase dimers, with the goal of developing a full kinetic mechanism for unwinding. DNA binding, ATP hydrolysis and DNA unwinding by RecBCD and RecBC helicases will also be examined mechanistically. Thermodynamic studies will be performed to understand the energetics of the binding of these helicases to DNA, and how DNA binding energy is used in DNA unwinding. We will also examine mutants of these enzymes, including constitutive dimers of Rep, and the mechanism by which MutL stimulates DNA unwinding by UvrD. These ensemble studies will be complemented by single molecule studies of DNA binding and unwinding and structural studies by x-ray crystallography.