Kinesin is a mechanoenzyme that drives microtubule-based intracellular organelle transport processes. RecBCD is a DNA helicase/nuclease that generates single-stranded DMA ends required for DMA repair by homologous recombination. Int is a tyrosine recombinase that catalyzes the DNA strand cleavage and ligation reactions needed to insert a bateriophage genome into the host by site-specific DNA recombination. All of these enzymes enzymes couple a free-energy-liberating chemical reaction (either nucleotide hydrolysis or protein-DNA binding) to free-energy-requiring mechanical processes that the enzyme must execute to perform its proper biological function. In all of these systems, we want to characterize the mechanical processes and to determine how these processes are coupled the chemical reactions that drive them. To do that, 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. Site-specific recombination functions in transcription regulation and other processes in both prokaryotes and eukaryotes. In all of these systems, we will investigate the functioning of these systems at the molecular level in order to learn more about the basic biology of how these systems maintain, or through disfunction fail to maintain, normal cellular operation.
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