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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Method to Extend Research in Time (MERIT) Award (R37)
Project #
Application #
Study Section
Special Emphasis Panel (NSS)
Program Officer
Gindhart, Joseph G
Project Start
Project End
Budget Start
Budget End
Support Year
Fiscal Year
Total Cost
Indirect Cost
Brandeis University
Schools of Arts and Sciences
United States
Zip Code
Anderson, Eric G; Hoskins, Aaron A (2014) Single molecule approaches for studying spliceosome assembly and catalysis. Methods Mol Biol 1126:217-41
Smith, Benjamin A; Daugherty-Clarke, Karen; Goode, Bruce L et al. (2013) Pathway of actin filament branch formation by Arp2/3 complex revealed by single-molecule imaging. Proc Natl Acad Sci U S A 110:1285-90
Smith, Benjamin A; Padrick, Shae B; Doolittle, Lynda K et al. (2013) Three-color single molecule imaging shows WASP detachment from Arp2/3 complex triggers actin filament branch formation. Elife 2:e01008
Breitsprecher, Dennis; Jaiswal, Richa; Bombardier, Jeffrey P et al. (2012) Rocket launcher mechanism of collaborative actin assembly defined by single-molecule imaging. Science 336:1164-8
Garcia, Hernan G; Sanchez, Alvaro; Boedicker, James Q et al. (2012) Operator sequence alters gene expression independently of transcription factor occupancy in bacteria. Cell Rep 2:150-61
Friedman, Larry J; Gelles, Jeff (2012) Mechanism of transcription initiation at an activator-dependent promoter defined by single-molecule observation. Cell 148:679-89
Sanchez, Alvaro; Garcia, Hernan G; Jones, Daniel et al. (2011) Effect of promoter architecture on the cell-to-cell variability in gene expression. PLoS Comput Biol 7:e1001100
Hoskins, Aaron A; Friedman, Larry J; Gallagher, Sarah S et al. (2011) Ordered and dynamic assembly of single spliceosomes. Science 331:1289-95
Ydenberg, Casey A; Smith, Benjamin A; Breitsprecher, Dennis et al. (2011) Cease-fire at the leading edge: new perspectives on actin filament branching, debranching, and cross-linking. Cytoskeleton (Hoboken) 68:596-602
Hoskins, Aaron A; Gelles, Jeff; Moore, Melissa J (2011) New insights into the spliceosome by single molecule fluorescence microscopy. Curr Opin Chem Biol 15:864-70

Showing the most recent 10 out of 17 publications