All ETDs from UAB

Advisory Committee Chair

Robert Cam Van Waardenburg

Advisory Committee Members

Robert Cam Van Waardenburg

Eddy S-H Yang

Karina J Yoon

Document Type

Thesis

Date of Award

2021

Degree Name by School

Master of Science in Biomedical Science (MSBMS) School of Engineering

Abstract

Tyrosyl-DNA phosphodiesterase 1 (Tdp1) removes adducts from DNA-ends, including damaged nucleotides and protein-DNA crosslinks such as DNA topoisomerases (Topo1 and Topo2) and Tdp1 itself. The N-terminal domain (NTD) of Tdp1 has been understudied, structurally unresolved, and seemingly dispensable for in vitro catalytic activity. However, our lab made the novel observation that the NTD is critical for Tdp1 cellular activity in removing Topo1-DNA covalent complexes (Topo1cc). Removal of the first 80 residues (yeast Tdp1 NTD) results in the loss of the toxic phenotype induced by full-length Tdp1 catalytic mutants. This suggests that in cells the NTD has a critical function in Tdp1 repair of Topo1cc. However, the role of Tdp1 and its NTD in the removal of Topo2cc is understudied. We hypothesize that Tdp1’s ability to repair Topo2cc is dependent on its NTD. To test this hypothesis, we generated yeast strains that reduce the efflux of FDA-approved Topo2 poison etoposide. We used previously characterized Tdp1 catalytic mutants to assess Tdp1 repair of Topo2cc with or without etoposide in colony formation assays. Compared to Topo1cc, Tdp1 only repairs etoposide-Topo2cc. Our preliminary results suggest that wild type Tdp1 is able to suppress the etoposide/Topo2cc-dependent Tdp1 induced toxicity. Moreover, based on our preliminary data, we observed that the removal of Tdp1 NTD results in the loss of the toxic phenotype induced by full-length Tdp1 catalytic mutants. Similarly, this suggests that the NTD is essential for the repair of etoposide-Topo2cc. In summary, we made the novel observation that Tdp1 can only repair drug stabilized Topo2cc, which is dependent on Tdp1 NTD, and that Tdp1 is able to hydrolyze 5’-phosphohystidyl linkage within a Tdp1cc.

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