Single-molecule studies of mechanical and helicase-catalyzed disruption of nucleic acid duplexes

Whitley, Kevin D

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Description

Title

Single-molecule studies of mechanical and helicase-catalyzed disruption of nucleic acid duplexes

Author(s)

Whitley, Kevin D

Issue Date

2017-04-20

Director of Research (if dissertation) or Advisor (if thesis)

Chemla, Yann R.

Doctoral Committee Chair(s)

Chemla, Yann R.

Committee Member(s)

• Aksimentiev, Oleksii
• Selvin, Paul R.
• Ha, Taekjip

Department of Study

School of Molecular & Cell Bio

Discipline

Biophysics & Computnl Biology

Degree Granting Institution

University of Illinois at Urbana-Champaign

Degree Name

Ph.D.

Degree Level

Dissertation

Keyword(s)

• Nucleic acids
• Optical tweezers
• Single-molecule
• Fluorescence
• Helicases

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.

Graduation Semester

2017-05

Type of Resource

text

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Copyright and License Information

Copyright 2017 Kevin Whitley

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