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Developing a Model System to Probe Biological Mechanisms of Post-Translational Modifications that Destabilize the Nucleosome

Beasley, Miranda L
http://orcid.org/0000-0002-5089-2073
http://rave.ohiolink.edu/etdc/view?acc_num=osu1408961013

Abstract Details

2014, Master of Science, Ohio State University, Biochemistry.
To develop a model system to probe biological cellular processes, we synthesized histones with four modifications along the histone-DNA interface: acetylation of lysines 115 and 122 of H3 (H3-K115, 122ac) in the dyad region and acetylation of lysines 77 and 79 of H4 (H4-K77, 79ac) in the loss of rDNA silencing (LRS) region. The objective of the current work is to utilize our model system containing modifications previously shown to perturb the nucleosome in order to probe various biological processes such that we can gain new knowledge and insight into the specific mechanisms associated with that process. Two peptides were synthesized, histone H3 (110-135; K115ac, K122ac) and histone H4 (76-102; K77ac, K79ac). Expressed protein ligation was used to generate semi-synthetic proteins H3-K115, 122ac and H4-K77, 79ac. WT proteins were refolded into histone octamer and reconstituted into nucleosomes. As a proof of principle, reconstituted WT nucleosomes were utilized in the preliminary retrovirus integration assays with prototype foamy virus integrase (PFV IN) and a 300 base pair donor DNA molecule. PFV IN processes the U5 end of long terminal repeats and catalyzes the strand transfer of retroviral cDNA (donor DNA) into the host (target DNA), resulting in a donor-host hybrid. Our initial results show that PFV does not integrate into naked DNA, but has a preference for DNA wrapped into nucleosomes. Additionally, we show what appears to be concerted integration and half-site integration into donor DNA containing both dual U5 donor and single U5 donor ends. In Chapter 1, I will provide a review of histone proteins. This will include a brief background on histones and post-translational modifications (PTMs) and the methods used to study PTMs, focusing on the use of genetic studies and chemical ligation tools. In Chapter 2, I will first describe modifications that destabilize the nucleosome through work that we have performed in our lab. Next, I will describe the methods that were used to synthesize the semi-synthetic proteins (H3-K115, 122ac and H4-K77, 79ac). The last part of this chapter will include the results from preliminary integration assays with PFV IN.
Jennifer Ottesen (Advisor)
Thomas Magliery (Committee Member)
107 p.
Biochemistry
Histones; post-translational modifications; nucleosomes; retrovrial integration; prototype foamy virus

Recommended Citations

Citations

  • Beasley, M. L. (2014). Developing a Model System to Probe Biological Mechanisms of Post-Translational Modifications that Destabilize the Nucleosome [Master's thesis, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1408961013

    APA Style (7th edition)

  • Beasley, Miranda. Developing a Model System to Probe Biological Mechanisms of Post-Translational Modifications that Destabilize the Nucleosome. 2014. Ohio State University, Master's thesis. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1408961013.

    MLA Style (8th edition)

  • Beasley, Miranda. "Developing a Model System to Probe Biological Mechanisms of Post-Translational Modifications that Destabilize the Nucleosome." Master's thesis, Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1408961013

    Chicago Manual of Style (17th edition)

osu1408961013
120
© 2014, some rights reserved.Creative Commons License Developing a Model System to Probe Biological Mechanisms of Post-Translational Modifications that Destabilize the Nucleosome by Miranda L Beasley is licensed under a Creative Commons Attribution-NonCommercial-NoDerivs 3.0 Unported License. Based on a work at etd.ohiolink.edu.
This open access ETD is published by The Ohio State University and OhioLINK.