All ETDs from UAB

Advisory Committee Chair

Robert Cam Van Waardenburg

Advisory Committee Members

Mary-Ann Bjornsti

Charles N Falany

David Schneider

Joanne Murphy-Ulrich

Shih-Hsin "Eddy" Yang

Document Type

Dissertation

Date of Award

2017

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Tyrosyl-DNA phosphodiesterase I (Tdp1) is a conserved eukaryotic DNA repair enzyme that removes adducts from DNA-ends. This includes DNA topoisomerase I (Top1)-DNA covalent complexes reversibly stabilized by camptothecins (CPTs). Tdp1s two-step catalytic cycle requires the action of its nucleophilic histidine (Hisnuc) that detaches the adduct by forming a Tdp1-DNA adduct, and its general acid/base histidine (Hisgab) to activate water, thereby releasing Tdp1 from the DNA-end. Tdp1 expression is associated with genomic instability, cancer etiology, chemotherapeutic resistance, and SCAN1-neurodegeneration, via Tdp1HisgabArg catalytic mutant. Our lab has characterized substitutions of the catalytic histidines in both yeast and human cell models. These substitutions exhibit more stable Tdp1-DNA adducts (reduced catalytic activity) that reduces cell viability. Our results suggest that Tdp1 can be converted from a DNA repair enzyme into a cellular toxin, by stabilizing the enzyme-DNA adducts. A proof-of-principle experiment expressing Tdp1 mutants in stably- transfected human cells treated with topotecan supported this novel strategy. Studies within this thesis show that the N-terminal domain of Tdp1 is critical for Tdp1 cellular function; stimulates in vitro catalysis and the stability of Tdp1-DNA adducts. Among Tdp1 proteins, the N-terminal domain is poorly conserved in sequence and size. It is also structurally and functionally understudied. Nuclear expression of the N-terminally truncated proteins did not elicit the cytotoxicity induced by full-length enzymes. Moreover, in vitro analysis of catalytic activity revealed that the N-terminal domain stimulates catalytic activity by reducing the stability of the Tdp1-DNA reaction intermediate. Overall, these results revealed that the Tdp1 N-terminal domain plays a dynamic role during catalysis and Tdp1’s function in cells. It has also improved our understanding of how Tdp1 and Top1-DNA adducts might interact, which is essential to develop Tdp1 as a therapeutic target. Our proof-of-principle experiment supported the novel therapeutic strategy of turning the DNA repair enzyme into a cellular toxin, by stabilizing its enzyme-DNA reaction intermediate, similar to CPT stabilization of the Topo1-DNA adduct. To further support the development of Tdp1 as a therapeutic target it is important that we fully understand the mechanism of interaction between Tdp1 and its adducted DNA substrates, specifically the Top1-DNA adduct.

Share

COinS