The integrity of our DNA is constantly under threat from many internal and external factors. If the cell cannot properly protect the integrity of DNA, errors (lesions) in DNA may form which can lead to cancer. Most of these lesions serve as a roadblock to the protein, DNA polymerase (Pol), that replicates the DNA during cell division. The mechanism that is employed to read through these errors is called translesion DNA synthesis (TLS). During this process, a special class of DNA polymerases known as TLS DNA Pols that can tolerate and bypass the lesions in DNA are employed by the cell. The objective of this research is to synthesize and purify the human TLS DNA polymerase kappa (Pol k) and study the interaction of Pol k with DNA using optical tweezers. A purification protocol for Pol k was successfully developed and accomplished by consecutive chromatography using affinity, ion-exchange, and size-exclusion columns. This provided us with the necessary level of purification for studies at the single-molecule level. A dual-beam optical tweezers set-up that uses two counter-propagating lasers finely focused inside a flow-cell was used to trap a single DNA molecule. The trapped DNA was stretched in the presence of various concentrations of Pol k and in the absence of Pol k to obtain the force felt by DNA as a function of extension. The preliminary results suggest that Pol k can bind to both double-stranded and single-stranded DNA with slow binding kinetics in the order of seconds. To our knowledge, this is the first attempt to explore the binding of Pol k to DNA at the single-molecule level. Complete characterization of the binding with this technique will provide us with a better understanding of Pol k binding mechanism to DNA at the molecular level and help us develop therapeutics that target translesion synthesis.
Physics, Photonics, and Optical Engineering and Chemical Sciences
Dr. Thayaparan Paramanathan, Thesis Advisor
Dr. Samer Lone, Thesis Advisor
Dr. Alyssa Deline, Committee Member
Dr. Elif Demirbas, Committee Member
Watts, Joshua. (2022). Using Optical Tweezersto Probe DNA Polymerase Kappa’s Binding Mechanism to DNA. In BSU Honors Program Theses and Projects. Item 569. Available at: https://vc.bridgew.edu/honors_proj/569
Copyright © 2022 Joshua Watts