Kinesin is a mechanoenzyme that drives microtubule-based intracellular organelle transport processes. RecBCD is a DMAhelicase/nuclease that generates single-stranded DNA ends required for DNA repair by homologous recombination. Both enzymes couples a free-energy- liberating chemical reaction (the hydrolysis of ATP) to a cycle of mechanical processesthat move the enzyme molecule along its microtubule or DNA track. We want to characterize the cycle of mechanical processes by which these enzymes move and to determine how these processes are coupled to the reactions of ATP hydrolysis. To facilitate this work, we have developed and use single-molecule biophysics techniques that allow us to directly monitor nanometer-scale mechanical processes, domain movements, and chemical steps in single, isolated enzyme molecules using light microscope-based instruments. Intracellular organelle transport by kinesin and kinesin homologs plays an essential role in the physiology of eukaryotic cells. Its functions include transport of materials, chromosome and nuclear movements in mitosis/meiosis, and morphogenesis of membranous organelles. DNA repair by homologous recombination is also an essential cellular function that restarts broken replication forks to permit full replication of the cellular genome. To explore these functions at the molecular level, we will: 1) Test whether ATP hydrolysis by both head domains is essential for processive, high-duty-ratio movement by kinesin;2) Test whether the first catalytic turnover of a kinesin-microtubule complex is structurally identical to subsequent turnovers; 3) Characterize the force-dependent translocation step(s) in the catalytic cycle of the RecBCD helicase activity by measuring the force-velocity relationship of the enzyme;4) Measure the size of the unitary steps in the movement of RecBCD along duplex DNA.
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