Mechanistic Studies of Type II Topoisomerases and Topoisomerase-Targeted Agents

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Enter the text here that is the new abstract information for your application. This section must be no longer than 30 lines of text. Type II topoisomerases are ubiquitous enzymes that are required for proper chromosome structure and segregation and play important roles in DNA replication, transcription, and recombination. These enzymes relax DNA and remove knots and tangles from the genetic material by passing an intact double helix (transport segment) through a transient double-stranded break that they generate in a separate DNA segment (gate segment). Humans encode two closely related isoforms of the type II enzyme, topoisomerase IIα and topoisomerase IIβ. Topoisomerase IIα is essential for the survival of proliferating cells and topoisomerase IIβ plays critical roles during development. However, because these enzymes generate requisite double-stranded DNA breaks during their crucial catalytic functions, they assume a dual persona. Although essential to cell survival, they also pose an intrinsic threat to genomic integrity every time they act. Beyond their critical physiological functions, topoisomerase IIα and IIβ are the primary targets for some of the most active and widely prescribed drugs currently used for the treatment of human cancers. These agents kill cells by stabilizing covalent topoisomerase II-cleaved DNA complexes (cleavage complexes) that are normal, but fleeting, intermediates in the catalytic DNA strand passage reaction. When the resulting enzyme-associated DNA breaks are present in sufficient concentrations, they can trigger cell death pathways. Anticancer drugs that target type II enzymes are referred to as topoisomerase II poisons because they convert these indispensable enzymes to potent physiological toxins that generate DNA damage in treated cells. Although topoisomerase IIα and IIβ are important targets for cancer chemotherapy, evidence suggests that they also have the potential to trigger specific leukemias. A small percentage of cancer (and other) patients treated with topoisomerase II-targeted drugs eventually develop acute myeloid leukemias (AMLs) involving the MLL gene at chromosome band 11q23 or acute promyelocytic leukemias involving 15:17 translocations. The 11q23 chromosomal translocations also are seen in infant AMLs, and the risk of these leukemias rises ~3–fold when there is high maternal exposure during pregnancy to environmental and dietary topoisomerase II poisons. Despite the importance of type II topoisomerases to cell growth and cancer, we still have much to learn about how the human enzymes function and interact with DNA and anticancer drugs in vitro, in cells, and in vertebrate animals. Thus, the proposed aims are designed to further define the catalytic mechanism and DNA interactions of topoisomerase II and assess the mechanism by which novel topoisomerase II-targeted drugs and drug-DNA conjugates increase levels of enzyme-mediated DNA breaks in vitro, in cultured cells, and in vertebrate animals. The primary research models for this study will be human topoisomerase IIα and IIβ, cultured human cells, and Xenopus laevis extracts. Bacterial gyrase and topoisomerase IV also will be used to assess relationships between the mechanisms of action of drugs targeted to prokaryotic and eukaryotic type II enzymes.