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Single Molecule Analysis of DNA Interactions

Jones, Nathan, Jones
http://rave.ohiolink.edu/etdc/view?acc_num=osu1511959163350735

Abstract Details

2017, Doctor of Philosophy, Ohio State University, Biophysics.
Magnetic tweezers provides a versatile toolkit supporting the mechanistic investigation of DNA substrates and protein association. First, the fundamental concepts of magnetic force are explained in detail. A custom magnetic tweezers system is assembled and optimized using optical components. A DNA substrate capable of interacting with a surface and magnetic particle is constructed from smaller DNA pieces. A flow cell for single molecule experiments is diagramed, including functionalization of the flow cell surface. Finally, tests are performed measuring the lateral and axial resolution limits of the system. Retroviruses must integrate their linear viral cDNA into the host genome for a productive infection. Integration is catalyzed by the retrovirus-encoded integrase (IN), which forms a tetramer complex with the two viral cDNA ends (intasome), removes two 3’-nucleotides and catalyzes end-joining (strand transfer) of the recessed 3’-hydroxyls separated by 4-6 bp of the target DNA. Structures of prototype foamy virus (PFV) integrase tetramers suggest the two strand transfer events may not be coordinated. Here we have used multiple single molecule imaging systems to determine that the Prototype Foamy Virus (PFV) intasome searches for a target site by 1-dimensional (1D) rotational diffusion while in continuous contact with the target DNA. It then catalyzes the two-step strand transfer in 470 ms. The vast majority of PFV intasome search events on a target DNA were non-productive. These observations suggest that target site-selection is rate limiting during retroviral integration, identifying a separate IN function that might be pursued for therapeutic intervention. Eukaryotic telomeres consist of tandem repeats containing 3-4 guanine nucleotides (G-strand) that commonly terminate in a 100-1000 nt single stranded DNA (ssDNA) 3’-tail. Four adjacent G-strand ssDNA repeats characteristically fold into flattened 0.63 nm x 4.1 nm parallel G-quadruplex disc-like structures. We generated long human G-strand ssDNA (5’-TTAGGG-3’)n that formed ubiquitous G-quadruplex folds based on structure-specific fluorescent-dye exclusion. Electron microscopy revealed chains containing 5 nm and 8 nm bead-like particles linked by thin filaments. This G-strand ssDNA displayed initial stability to single molecule magnetic tweezers force extension that ultimately released in steps that were multiples ~28 nm at forces between 6-12 pN; well below the >20 pN required to unravel G-quadruplexes. RAD51 binding with and without ATP distinctively altered the G-strand force extension. These observations indicate that at least four G-quadruplex elements may condense into a higher-order complex stabilized by surplus G-strand repeat loop interactions, providing additional structural layers to chromosome ends.
Richard Fishel (Advisor)
Ralf Bundschuh (Committee Member)
Marcos Sotomayor (Committee Member)
Charles Bell (Committee Member)
138 p.
Biophysics
Molecule Analysis; DNA Interactions; Biophysics

Recommended Citations

Citations

  • Jones, Jones, N. (2017). Single Molecule Analysis of DNA Interactions [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1511959163350735

    APA Style (7th edition)

  • Jones, Jones, Nathan. Single Molecule Analysis of DNA Interactions. 2017. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1511959163350735.

    MLA Style (8th edition)

  • Jones, Jones, Nathan. "Single Molecule Analysis of DNA Interactions." Doctoral dissertation, Ohio State University, 2017. http://rave.ohiolink.edu/etdc/view?acc_num=osu1511959163350735

    Chicago Manual of Style (17th edition)

osu1511959163350735
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© 2017, all rights reserved.
This open access ETD is published by The Ohio State University and OhioLINK.