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Title:Single-molecule studies of mechanical and helicase-catalyzed disruption of nucleic acid duplexes
Author(s):Whitley, Kevin D
Director of Research:Chemla, Yann R.
Doctoral Committee Chair(s):Chemla, Yann R.
Doctoral Committee Member(s):Aksimentiev, Oleksii; Selvin, Paul R.; Ha, Taekjip
Department / Program:School of Molecular & Cell Bio
Discipline:Biophysics & Computnl Biology
Degree Granting Institution:University of Illinois at Urbana-Champaign
Subject(s):Nucleic acids
Optical tweezers
Abstract:Nucleic acids (e.g. DNA, RNA) are subjected to numerous twisting, bending, and stretching forces within cells, and enzymes process them in a variety of ways. The behavior of nucleic acids in response to applied forces and enzymatic activity is therefore necessary for a fundamental understanding of biology. Furthermore, a detailed knowledge of nucleic acids and the enzymes that process them has fueled advances in bio- and nano-technology. In this thesis, we focus on two main systems: the elastic behavior of ultrashort nucleic acids and the activity of E. coli UvrD helicase. First, we use a hybrid instrument combining high-resolution optical tweezers with single-fluorophore sensitivity to observe the hybridization of ultrashort (<15 nt) DNA and RNA oligonucleotides under tension, one molecule at a time. We quantify the effect of tension on the rates of hybridization, and in doing so determine the elastic behavior of the transition state for the reaction. We then investigate the elasticity of the ultrashort oligonucleotides by observing the change in extension that takes place during hybridization. Our results enable us to produce a model describing the shear-induced fraying of base-pairs in a nucleic acid duplex. We then use similar single-molecule techniques to characterize E. coli UvrD helicase. First, we investigate UvrD’s stepping dynamics by directly observing individual motor steps of the protein. Then, we examine the factors influencing the ability of UvrD to switch between unzipping and re-zipping behaviors. Finally, we place UvrD in its biological context by observing the effect of its interactions with an accessory protein in DNA mismatch repair.
Issue Date:2017-04-20
Rights Information:Copyright 2017 Kevin Whitley
Date Available in IDEALS:2017-08-10
Date Deposited:2017-05

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