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An Investigation of a G-Quadruplex and Its Interactions with Human Replication Protein A at the Single Molecule Level

Malcolm, Dominic W.
http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335812645

Abstract Details

2012, BS, Kent State University, College of Arts and Sciences / Department of Physics.
A single molecule FRET study was performed to gather structural and dynamic information about a G-Quadruplex (GQ) in the presence of potassium ions (K+) and Replication Protein A (RPA), a predominant single strand DNA binding protein. A GQ is a non-canonical DNA structure that forms in guanosine (G) rich sequences. The structure is composed of stacked tetrads, each formed by four G residues, and loops of unstructured ssDNA. This study concentrates on characterization of a three-layer GQ with loops that are three nucleotides long each. The results confirm the formation of the GQ structure, while revealing nearly 100% folding at the physiological concentration of K+ (150 mM). It was also found that RPA-mediated unfolding dominates folding by K+ under physiological conditions, giving some insight into this structure's physiological viability. The timescales of transitioning from an unfolded single strand structure to a folded GQ, along with the transition from a folded GQ to an RPA-bound unfolded structure were also measured in this single molecule assay. The transition from unfolded to folded GQ structure was measured to be 0.35±0.10 seconds, while the transition from a folded GQ to an RPA-bound unfolded structure was 0.28±0.10 seconds. These results suggest that the folding and unfolding dynamics of this structure take place on a similar timescale. The rest of this study compares the results from this specific GQ structure to results gathered from the same assays on other GQ structures with varying number of layers and loop lengths to draw conclusions about these structures’ physiological relevance.
Hamza Balci, PhD (Advisor)
Elizabeth Mann, PhD (Committee Member)
Soumitra Basu, PhD (Committee Member)
Natasha Levinson, PhD (Committee Member)
69 p.
Biology; Biophysics; Optics; Physics
FRET; single molecule; G-Quadruplex; GQ; Replication Protein A; RPA; smFRET; DNA; Biophysics; Total Internal Reflection Fluorescence Microscopy; TIRFM

Recommended Citations

Citations

  • Malcolm, D. W. (2012). An Investigation of a G-Quadruplex and Its Interactions with Human Replication Protein A at the Single Molecule Level [Undergraduate thesis, Kent State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335812645

    APA Style (7th edition)

  • Malcolm, Dominic. An Investigation of a G-Quadruplex and Its Interactions with Human Replication Protein A at the Single Molecule Level. 2012. Kent State University, Undergraduate thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335812645.

    MLA Style (8th edition)

  • Malcolm, Dominic. "An Investigation of a G-Quadruplex and Its Interactions with Human Replication Protein A at the Single Molecule Level." Undergraduate thesis, Kent State University, 2012. http://rave.ohiolink.edu/etdc/view?acc_num=ksuhonors1335812645

    Chicago Manual of Style (17th edition)

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© 2012, all rights reserved.
This open access ETD is published by Kent State University Honors College and OhioLINK.