Date of Award

12-2007

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Interdisciplinary Program

Research Advisor

Mary-Ann Bjornsti, Ph.D.

Committee

Terrance G. Cooper, Ph.D. Katsumi Kitagawa, Ph.D. Martine F. Roussel, Ph.D. Brenda A. Schulman, Ph.D.

Keywords

DNA topoisomerase I, camptothecin, enzyme architecture, domain flexibility, drug sensitivity, structural alteration

Abstract

DNA topoisomerase I (Top1) is a highly conserved enzyme composed of four domains: a positively charged N-terminus; a DNA binding/core domain that circumscribes duplex DNA; a non-conserved linker domain; and a C-terminal/catalytic domain. Top1 catalyzes changes in DNA topology by transient cleavage of a single DNA strand and the concomitant formation of a phosphotyrosyl linkage between the enzyme and the 3’ DNA end. This covalent Top1-DNA complex is the binding site for camptothecin (CPT), which selectively inhibits religation of the cleaved DNA strand. CPT binding stabilizes the covalent complex, while the collision of replication forks with CPT-Top1-DNA adducts produces DNA lesions that induce cell death. Mutation of conserved residues in close proximity to the active site tyrosine, Tyr727 in yeast Top1 (yTop1) and Tyr 723 in human Top1 (hTop1), alters DNA cleavage-religation equilibrium, inducing drug independent lethality. yTop1T722 A mimics the action of CPT and decreases the rate of religation, while yTop1N726 H increases the rate of cleavage. Even though both mutants increase the stabilization of the Top1-DNA covalent complexes, it is evident that distinct cytotoxic lesions are formed by the different responses induced by low level expression of these self-poisoning enzymes in isogenic yeast strains defective for the Rad9 DNA damage checkpoint, processive DNA replication, or ubiquitin-mediated proteolysis. The core and C-terminal domains are connected by extended α-helices (linker domain), which position the active site Tyr within the catalytic pocket. The physical connection of the linker domain with the Top1 clamp as well as linker flexibility v affect enzyme sensitivity to CPT. Crystallographic data reveal a conserved Gly residue (located at the juncture between the flexible linker and C-terminal domains) that lies at one end of a short α-helix, which extends to the active site Tyr covalently linked to the DNA. However, in the presence of drug, the linker is rigid and this α-helix extends to include Gly and the preceding Leu, suggesting a dynamic interplay between active site α- helical structure and linker flexibility. In vitro and in vivo analyses of Gly 721 mutations in yTop1 demonstrate that charge and α-helical propensity of amino acid side chains at position 721 impact active site architecture within the enzyme-DNA complex. In combination with the crystallographic data, these results implicate the conserved Gly 721 as a flexible hinge within the active site of Top1 that enables linker flexibility and the structural alterations that accompany drug binding of the covalent enzyme-DNA complex. In vitro studies of the corresponding human mutant, hTop1G717 D, showed that this mechanism of increased sensitivity is conserved. However, by expressing hTop1G717 D in yeast cells the increased CPT sensitivity was not recapitulated. To determine the contribution of the divergent N-terminal and linker domains to enzyme activity and in vivo CPT sensitivity, a series of chimeric enzymes, involving reciprocal swaps of the yeast and human N-termini and linkers, were generated. The results demonstrate that the conserved core and C-terminal domains dictate the intrinsic enzyme sensitivity to CPT, while it is the functional interactions of the N-terminal and linker domains that regulate enzyme activity in vivo.

DOI

10.21007/etd.cghs.2007.0333

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