TY - THES
AU - Stephen Okoniewski
AB - In this thesis, I detail the work I have done in studying both force-activated DNA substrates and the RecQ DNA helicase from E. coli. The first half of the document introduces some background for these topics as well as the experimental methods used, with a focus on my high-precision single-molecule optical trap. In the chapter on force-activated DNA substrates, I explain the rationale for developing them, prior strides towards completing them, and then our work. Multiple substrate types are explored and validated, and several single-molecule helicase assays are done as proof of construct efficacy. The last chapter deals with my research on the single-molecule unwinding behavior of the RecQ helicase. The main focus of the chapter is optimizing my assay conditions so I can resolve single base-pair steps of RecQ, in order to better understand its fundamental stepping mechanics and see whether the kinetic models posed in previous papers are correct. I end the thesis by discussing some novel interactions between RecQ and its DNA substrate which lead to unexpected unwinding behaviors.
BT - Department of Physics
CY - Boulder
DA - 2020-06
N2 - In this thesis, I detail the work I have done in studying both force-activated DNA substrates and the RecQ DNA helicase from E. coli. The first half of the document introduces some background for these topics as well as the experimental methods used, with a focus on my high-precision single-molecule optical trap. In the chapter on force-activated DNA substrates, I explain the rationale for developing them, prior strides towards completing them, and then our work. Multiple substrate types are explored and validated, and several single-molecule helicase assays are done as proof of construct efficacy. The last chapter deals with my research on the single-molecule unwinding behavior of the RecQ helicase. The main focus of the chapter is optimizing my assay conditions so I can resolve single base-pair steps of RecQ, in order to better understand its fundamental stepping mechanics and see whether the kinetic models posed in previous papers are correct. I end the thesis by discussing some novel interactions between RecQ and its DNA substrate which lead to unexpected unwinding behaviors.
PB - University of Colorado Boulder
PP - Boulder
PY - 2020
EP - 178
T2 - Department of Physics
TI - A Single-Molecule Optical Trapping Assay: Measuring E. coli RecQ Helicase Motion Using Force-Activated DNA Substrates
VL - Ph.D.
ER -