Quinone Metabolites of Environmental Toxins Poison Topoisomerase IIalpha

Abstract

Topoisomerases play critical roles in maintaining DNA topology during cellular processes such as DNA replication in eukaryotes. Movement of the replication machinery through the double helix induces positive supercoiling ahead of the fork and precatenanes behind it. Because topoisomerase I and II create transient single- and double-stranded DNA breaks, respectively, it has been assumed that topoisomerase I relaxes the positive supercoils while topoisomerase II resolves precatenanes. In contrast to this proposed segregation of function, models for anticancer drug action place topoisomerase II ahead of replication forks. This discrepancy raises the question of whether eukaryotic type II topoisomerases have normal physiological functions ahead of DNA tracking systems. If so, then positively supercoiled DNA might be the preferred substrate for human topoisomerase IIá, the isoform involved in replicative processes. Therefore, the work described in this dissertation compared the activities of human topoisomerases on positively and negatively supercoiled DNA in the absence and presence of anticancer drugs, and explored the mechanisms by which topoisomerase II recognizes DNA supercoil geometry. First, this work characterized the abilities of human topoisomerase IIá and â to relax positively and negatively supercoiled DNA. Topoisomerase IIá, but not â, displayed characteristics that suggest it has the potential to relieve torsional stress ahead of approaching DNA tracking systems efficiently and safely. Second, this work examined the effects of positive DNA supercoiling on topoisomerase-mediated DNA cleavage and response to anticancer agents. Results indicate that DNA supercoil geometry has a profound influence on topoisomerase II-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either type II enzyme. Lastly, this work explored the mechanism by which topoisomerase II recognizes DNA supercoil geometry. Results suggest that the enzyme recognizes supercoil geometry in a bimodal fashion that involves elements in the N-terminal or central domain for cleavage and the variable C-terminal domain for relaxation. This ability has implications for the catalytic function of topoisomerase II and may account for some of the differences in the physiological roles played by distinct type II enzymes.