Monitoring of DNA Polymerase Theta Movements Through FRET Analysis

Citation data:

Honors Projects Overview

Publication Year:
2018
Usage 29
Downloads 20
Abstract Views 9
Repository URL:
https://digitalcommons.ric.edu/honors_projects/146; https://digitalcommons.ric.edu/cgi/viewcontent.cgi?article=1148&context=honors_projects
Author(s):
Rebelo, Ashley
Publisher(s):
Digital Commons @ RIC
Tags:
biochemistry; FRET; polymerase; DNA repair; Fluorescence Resonance Energy Transfer; FRET;; Cell and Developmental Biology; Physical Sciences and Mathematics
artifact description
DNA polymerases are enzymes used for DNA replication during cell division and can be specialized for DNA repair. DNA Polymerase Theta (Pol θ) is the predominant polymerase involved in alternative double-stranded break repair and is upregulated in breast cancer. It is errorprone as it does not accurately match the nucleotide on a DNA template with the correct complementary base. This inaccuracy affects the overall fidelity of the enzyme, a biochemical process that looks at the ability of a polymerase to “read” the template DNA and select the right nucleotide before polymerization. Polymerization for most DNA polymerases involves a global conformational change, specifically of the fingers domain, during insertion of a nucleotide after selecting it into the DNA strand in the active site. If this selection step is compromised, it could lead to the insertion of a non-complementary nucleotide, which could lead to mutations. The fingers domain is hypothesized to move closer to the active site during this process before release of the extended product, where the fingers domain returns to its original conformation. These results have been previously observed in high-fidelity polymerases β and Klenow. Pol β is involved in BER, and the Klenow fragment synthesizes DNA in E.coli. In both studies, the enzyme adopts a closed conformation with correct nucleotide. This study aims to elucidate the mechanism of Pol θ during DNA repair, and how the global movements of this enzyme affects fidelity. Using Fluorescence Resonance Energy Transfer (FRET), we internally fluorescently labeled the fingers domain of Pol θ to observe its interaction with DNA and an incoming nucleotide. Preliminary results suggest that only in the presence of correct nucleotide does Pol θ experience a global conformational change during polymerization to ensure correct matching, suggesting nucleotide selection by Pol θ is monitored in an open conformation and only when the correct pair is formed does polymerization proceed. Our FRET system can be used to further understand the fidelity of Pol θ and how it can lead to cancer