Investigation into the Structure and Function of Tyrosyl-DNA Phosphodiesterase I's N-Terminal Domain
Advisory Committee Chair
Robert Van Waardenburg
Advisory Committee Members
Stephen Aller
David Schneider
Eddy Yang
Karina Yoon
Document Type
Dissertation
Date of Award
2023
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Tyrosyl-DNA phosphodiesterase I (Tdp1) is a eukaryotic DNA repair enzyme that removes adducts from DNA ends via hydrolysis of a phosphodiester bond. These adducts consist of damaged nucleotides or trapped protein-DNA complexes. Tdp1 hydrolyzes the phosphodiester bond utilizing two catalytic histidines and forming a Tdp1-DNA covalent intermediate. Mutations of these catalytic histidines have been shown to increase the stability of the Tdp1-DNA covalent intermediate and cause Topoisomerase I dependent toxicity in cells. We use these toxic Tdp1 mutant enzymes to study the function of Tdp1’s N-terminal domain (NTD). Full-length Tdp1 has proven difficult to purify due to degradation of the NTD, and early reports involving full-length and NTD truncated Tdp1 suggested this domain to be dispensable for Tdp1 activity. Consequently, the NTD remained structurally unresolved and understudied in the field. In contrast to previous ideas in the field, our studies demonstrate that Tdp1’s NTD plays a critical role in the protein’s cellular and catalytic function. The removal of Tdp1’s NTD resulted in decreased catalytic activity in Tdp1 catalytic mutants in vitro and the loss of the toxic phenotype presented by their full-length counterparts in vivo. In the process of verifying Tdp1 protein expression and localization in our cellular experiments, we noted iv unexplained variability in Tdp1 protein levels in our cellular fractionation samples. We determined that a protease in Zymolyase, an enzyme mixture used to digest yeast cell walls, was responsible for degrading Tdp1 in these samples. Furthermore, we discovered that pre-incubation of the Zymolyase solution with PMSF protected Tdp1 from Zymolyase related degradation. Lastly, we provided an initial investigation of yeast Tdp1’s NTD by creating computationally generated prediction models of its structure. These models suggested that yeast Tdp1’s NTD is a flexible yet structured entity capable of adopting multiple conformations. Based on our results and available data, we hypothesize that Tdp1’s NTD is responsible for interacting with the DNA tail that extends from Tdp1’s catalytic core and stabilizes the substrate to facilitate efficient catalysis, as well as partaking in important protein-protein interactions that drive adduct resolution in cells.
Recommended Citation
Brettrager, Evan J., "Investigation into the Structure and Function of Tyrosyl-DNA Phosphodiesterase I's N-Terminal Domain" (2023). All ETDs from UAB. 444.
https://digitalcommons.library.uab.edu/etd-collection/444
