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 IIa and
topoisomerase IIß. Topoisomerase IIa is essential for the survival of proliferating cells and topoisomerase IIß
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 IIa and IIß 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 IIa and IIß are important targets for cancer chemotherapy, they also have the
potential to trigger specific leukemias. For example, a small percentage of patients with cancer or multiple
sclerosis who are treated with the topoisomerase II-targeted drug mitoxantrone go on to develop acute
promyelocytic leukemias (APLs) with 15:17 translocations.
Despite the importance of type II topoisomerases in cell growth and cancer, we still have much to learn about
how the human enzymes function and interact with DNA and anticancer drugs. Thus, this proposal will further
define the catalytic mechanism of type II topoisomerases and examine how enzyme activity is regulated in the
cell. It also will define mechanisms by which established and novel topoisomerase II-targeted agents, environ-
mental chemicals, and natural products increase levels of enzyme-mediated DNA breaks or inhibit enzyme
activity and determine the cellular consequences of topoisomerase II poisons. The primary research models for
this study will be human topoisomerase IIa and IIß, cultured human cells, and Xenopus laevis egg extracts.
Gyrase and topoisomerase IV from Escherichia coli and Bacillus anthracis will be used as counterpoints to
mechanistic experiments and Mycobacterium tuberculosis gyrase will be used to assess relationships between
the mechanisms of action of drugs targeted to prokaryotic and eukaryotic type II enzymes.