All ETDs from UAB

Advisory Committee Chair

Robert Cam Van Waardenburg

Advisory Committee Members

Charles N Falany

Mary-Ann Bjornsti

David Bedwell

David Graves

Document Type

Dissertation

Date of Award

2013

Degree Name by School

Doctor of Philosophy (PhD) Heersink School of Medicine

Abstract

Tyrosyl-DNA phosphodiesterase I (Tdp1) functions to remove 3'phospho-DNA adducts, such as eukaryotic DNA Topoisomerase I (Top1) covalently bound to the DNA by a 3'phospho-tyrosyl bond. As member of the Phospholipase D superfamily Tdp1 contains two HxK(n)N motifs, each of which provide a catalytic histidine: one functioning as a nucleophile to bind the 3'phosphate and the other as a general acid/base to hydrolyze Tdp1's 3'phospho-histidyl bond. Mutation of the general acid/base catalytic histidine (Hisgab) to arginine has been implicated in the autosomal recessive neurodegenerative disease SCAN1. Further study of the yeast enzyme revealed that substitution of Hisgab in yeast Tdp1 to small and/or non-reactive residues induced a Top1-dependent cytotoxicity more severe than the SCAN1-associated mutation, whereas substitution of Hisgab to lysine produced a wild type phenotype. The phenotype induced by the Tdp1 mutants, as well as their catalytic activity in vitro, depended upon both the size and chemistry of the residue replacing Hisgab. The reportedly inactive Hisnuc-alanine mutant induced cytotoxicity when expressed in yeast or human cells and showed catalytic activity in vitro, whereas Hisnuc-phenylalanine was inactive and did not induce cytotoxicity. Mutational analysis revealed that the Hisnuc-alanine-induced toxicity depends upon an adjacent conserved histidine. We posit that this conserved histidine functions as an alternative nucleophile when Hisnuc is substituted with a small, non-reactive residue. The toxicity and reduction in catalytic activity was similar for both yeast and human Tdp1 mutants, demonstrating that the catalytic mechanism and cellular response to dysregulation of Tdp1 is conserved from yeast to human. Our observations support the notion that dysregulation of Tdp1's catalytic mechanism causes stabilization of the Tdp1-DNA covalent reaction intermediate that induces cytotoxicity. This provides the basis for a novel therapeutic approach of targeting Tdp1 by utilizing small molecules to stabilize the covalent Tdp1-DNA complex, thus transforming this DNA repair enzyme into a cellular toxin for anti-cancer therapy.

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