@phdthesis{12645,
  author = {Ashley Carter},
  title = {A Precision Optical Trapping Assay: Measuring The Conformational Dynamics of Single E. Coli RecBCD Helicases},
  abstract = {Helicases are enzymes that unwind double-stranded nucleic acids [e.g., deoxyri- bonucleic acid (DNA)]. Unwinding occurs by converting chemical energy [e.g., adenosine triphosphate (ATP) hydrolysis] into a translocation-induced unwinding event, although the exact mechanism of how ATP is coupled to unwinding along DNA is unknown. The first step in understanding this mechanism is to determine the step size of movement. Structural data suggests that the mechanical step size of unwinding is one base-pair (bp) for each ATP molecule hydrolyzed, while biochemical data suggests that the rate-limited kinetic step size for unwinding is multiple base-pairs (∼4 bp). To address this question, we directly measured the translocation step size of a helicase, the E. coli RecBCD heli-case, using a single molecule assay. Previous single-molecule measurements of RecBCD failed to resolve steps due to limited resolution (∼6 bp). We increased resolution to 1 bp in a precision optical trapping assay by actively stabilizing the microscope and reducing multiple types of trap laser noise. With this precision assay, we observed both forwards and backwards steps that were variable in size and had an average step size of 4.4 bp. The variability and multiple base-pair step size are in part due to ATP-independent, ±3 bp conformational dynamics in the RecBCD-DNA complex. Thus, our results support a mechanism in which the RecBCD helicase has a variable step size; the average of which quantitatively agrees with the previously determined kinetic step size.},
  year = {2008},
  journal = {Department of Physics},
  volume = {Ph.D.},
  pages = {246},
  month = {2008-06},
  publisher = {University of Colorado Boulder},
  address = {Boulder},
}