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Structural Studies of DNA Repeats Implicated in Cancer

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dc.contributor.advisor Yatsunyk, Liliya A.
dc.contributor.author Jordan, Deondre
dc.date.accessioned 2019-07-12T14:20:10Z
dc.date.available 2019-07-12T14:20:10Z
dc.date.issued 2019
dc.identifier.uri http://hdl.handle.net/10066/21535
dc.description.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. en_US
dc.description.sponsorship Swarthmore College. Dept. of Chemistry & Biochemistry en_US
dc.language.iso en en_US
dc.rights Full copyright to this work is retained by the student author. It may only be used for non-commercial, research, and educational purposes. All other uses are restricted.
dc.title Structural Studies of DNA Repeats Implicated in Cancer en_US
dc.rights.access No restrictions en_US


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