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Single-Molecule Studies of DNA Motor Proteins Using Dual-Trap Optical Tweezers

  • Author(s): Deltoro, Damian Jorge
  • Advisor(s): Smith, Douglas E
  • et al.
Abstract

In this dissertation we take advantage of the real-time manipulating, high force generating, and small displacement measuring abilities of dual-trap optical tweezers to investigate properties and mechanisms of motor enzymes that act on DNA, including the DNA translocation motor of bacteriophage lambda and the proposed ATP-driven annealing helicase HARP. Through a combined approach of genetic mutagenesis, biochemical analysis, and single-molecule analysis we reveal potential functional roles of the residues residing in the ATP phosphate binding motif (Walker A) of phage lambda’s large terminase subunit. Direct DNA packaging measurements of phages with mutated residues within this motif reveal a range of impaired translocation phenotypes. Analysis of the slipping and pausing exhibited by mutant enzymes suggests that residues A78, R79, and V80 mediate coupling between ATP binding/hydrolysis and DNA binding/translocation while implicating residues A78, R79, V80, and G81 in proper ATP alignment for hydrolysis. The combined findings of each analysis also implicate residue R79 in ADP release and triggering ATP hydrolysis. In other studies, initial investigation into the mechanism of termination of DNA packaging in phage lambda provides evidence against a velocity-monitoring model. A higher resolution optical tweezers system was designed and constructed and a new and improved calibration method was developed and applied to initial high-resolution measurements of DNA translocation. The use of environmental and measurement noise reduction techniques and improved optical alignment procedures have yielded tweezers with approximately nanometer spatial resolution. The calibration technique, which optimizes fits of the worm-like chain model to DNA force-extension curves, yields calibration with at least a 7-fold improvement in accuracy over our prior method. The use of these tools in measuring lambda packaging yield preliminary evidence for discrete translocation steps. Lastly, tweezer measurements of HARP interactions with forked DNA provide supporting evidence that HARP is a forked-DNA binding protein that resists DNA unzipping and is not an unwinding helicase. In addition preliminary measurements suggest that this protein forcibly reanneals DNA, prevents reannealing, resists high forces, slows reannealing in an ATP dependent manner, and is recruited to bind to forked DNA with replication protein A coated single-stranded DNA.

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