Structural Studies of DNA Repeats Implicated in Cancer
Jordan, Deondre
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.
↧