Purification of PCBP2 and its Effect on the Formation of DNA i-Motifs and Secondary Structures

Author

Nathan ReddenFollow

Date of Award

Spring 5-2-2021

Document Type

Undergraduate Thesis

Department

Chemistry and Biochemistry

First Advisor

Randy Wadkins

Second Advisor

Saumen Chakraborty

Third Advisor

Susan Pedigo

Relational Format

Dissertation/Thesis

Abstract

Several secondary, non-B forms of DNA are associated with the promoter regions of many oncogenes, including the intensely studied c-MYC gene that codes for a transcription factor. Two such secondary structures, G-quadruplexes, and i-motifs, have become topics of interest due to their prevalence within oncogenic promoters, as well as their potential accessibility for drug targeting. B-form DNA can adopt alternative structures under certain conditions. These alternative structures are favored when there is an asymmetric distribution of guanosine and cytosine on complimentary DNA strands. The strand rich in guanosine is able to form a quadruple stranded DNA complex termed a G-quadruplex. The complementary cytosine-rich strand is also able to form a quadruple stranded DNA construct, known as an i-motif. Duplexes are more stable, thermodynamically, than the secondary structures that are formed from single stranded DNA. Therefore, an input of energy is required for DNA to form G-quadruplexes and i-motifs, resulting in higher ΔGo of formation than duplex DNA and an overwhelmingly endergonic process. Acidic conditions are typically required for the stabilization of i-motifs due to protonation of cytosines

that allows for three hydrogen bonds in C·C mismatches. However, crowding agents such as polyethylene glycols and dextrans can shift the pKa values of i-motifs to near physiological

conditions. Recent studies have also indicated that certain DNA strands such as SNORD112 and DUX4L22 are able to form i-motifs in near physiological conditions without the need for crowding agents, generating great interest in these DNA strands.

It is currently estimated that the amount of energy required for the opening of double stranded DNA and it’s conversion to 4-stranded structures is roughly 20-30 kcal/mol. The sources of this additional energy required to form i-motifs from duplex DNA are not entirely known. Recent research completed by a graduate student at the University of Mississippi has indicated that proteins binding to DNA i-motifs may contribute toward formation of i-motifs by providing a part of the required energy contribution. To estimate the energetic contributions of protein binding to the formation of i-motifs, Poly Cytosine Binding Protein 2 (PCBP2) was used as a model. PCBP2 is a protein that is known to bind to cytosine rich DNA. PCBP2 was an ideal candidate for this research, as PCBP2 is active in the acidic conditions necessary to induce i-motif formation in certain strands of DNA. Therefore, it is important to purify the protein, and improve upon the purification process to further determine the protein’s effect on the formation of i-motifs. PCBP2 is also homologous to the human hnRNP K protein, which is responsible for binding to pre- messenger RNA, and is a component of heterogeneous ribonucleoprotein particles2. It should be noted that hnRNP K is not a suitable candidate for the purposes of this research as hnRNP K is not active in the acidic conditions necessary for i-motif formation. Due to the COVID-19 pandemic and laboratory related constraints, purification of the PCBP2 protein and its DNA-binding KHIII domain was not fully completed. Therefore, previous studies are cited throughout this thesis to provide insight into relevant data that could not be gathered, while the gathered data that was accomplished for this thesis and its relevancy will also be discussed.

Recommended Citation

Redden, Nathan, "Purification of PCBP2 and its Effect on the Formation of DNA i-Motifs and Secondary Structures" (2021). Honors Theses. 1920.
https://egrove.olemiss.edu/hon_thesis/1920

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