Date of Award

Spring 2019

Document Type

Thesis

Terms of Use

© 2019 Deondre Jordan. All rights reserved. This work is freely available courtesy of the author. It may only be used for non-commercial, educational, and research purposes. For all other uses, including reproduction and distribution, please contact the copyright holder.

Degree Name

Bachelor of Arts

Department

Chemistry & Biochemistry Department

First Advisor

Liliya A. Yatsunyk

Abstract

DNA replication is an essential process for the survival of all organisms. Errors can occur during the process that leads to replication stress, which can promote genomic instability, a hallmark of cancer. The Brown lab conducted a genome wide study to identify sites and sequences linked to replication stress. Tandem DNA repeats comprised the sequences identified in this study. One particular DNA repeat (CAGAGG)ₙ stood out due to being highly associated with replication stress. Repeats such as (CAGAGG)ₙ are believed to fold into fold into intramolecular structures that physically block DNA polymerase during replication. Understanding the structures of these problematic DNA repeats may shed light on the mechanism behind replication stress and encourage the design of therapeutics to treat them. This works starts with biophysical characterization of identified DNA repeats associated with replication stress. This study highlighted (CAGAGG)ₙ as one of the most stable, well-folded, and spectroscopically unique repeats. Given the biological and biophysical data, we focused our efforts on elucidating the secondary structure of (CAGAGG)ₙ. Based on previous biophysical characterization in the Yatsunyk laboratory, a hypothetical model was designed for the smallest stable structure formed by the DNA repeat. The model is a tetrastranded monomer comprised of two-stacked GCGC tetrads connected by three lateral AGAG loops in an antiparallel fashion. The rest of the work presented here focuses on testing and refining the model for (CAGAGG)ₙ. To shed light on the secondary structure of (CAGAGG)ₙ, we conducted a ligand screen with over 20 known nucleic acid binders. If the DNA repeat interacts with a class of ligands specific for particular DNA structures (such as B-DNA), we can gain further structural information. Secondly, if a binder stabilizes the unique fold of (CAGAGG)ₙ, it can be used a co-crystallization agent to produce high quality crystals for structure determination via X-ray crystallography. This study showed (CAGAGG)ₙ interacts mostly with ligands specific for G-Quadruplex DNA. We also discovered Methylene Blue provided moderate stability to the DNA repeat. This ligand screen was extended using a Small Molecule Microarray assay, where 7042 ligands were simultaneously screened with (CAGAGG)ₙ. Biophysical characterization of the interactions of (CAGAGG)ₙ with some of the best hits from this assay showed one binder worth further study. To test our model for (CAGAGG)ₙ, we designed a variety of structural studied based on mutations. Some of these studies include: single point mutations, double point mutations, addition and removal of GCGC tetrads, loop length variations, and substitution of adenine with 2-amino purine. These studies showed the core was crucial for the stability, while the core and the loops together maintain the structural integrity. The loops proved to be crucial for the structure formation where the minimum loop length for the unique (CAGAGG)ₙ fold is three nucleotides. Overall, our biophysical studies corroborated our hypothetical model and shed light on the importance of the loops for the unique structure.

Comments

The research in this thesis is related to the following article: "Genome-wide Identification of Structure-Forming Repeats as Principal Sites of Fork Collapse upon ATR Inhibition."

Download

Included in

Chemistry Commons

Share

COinS