Graduate Student Vanderbilt University Nashville, Tennessee, United States
Jeffrey Jian (Vanderbilt University School of Medicine)| Jo Ann Byl (Vanderbilt University School of Medicine)| Neil Osheroff (Vanderbilt University School of Medicine , Vanderbilt University School of Medicine, VA Tennessee Valley Healthcare System)
Type II topoisomerases are enzymes that regulate DNA supercoiling and remove knots and tangles from the genome. Humans encode two isoforms of the type II enzyme, topoisomerases IIɑ and IIβ. Type II topoisomerases function by passing an intact double helix through a transient double-stranded break that they generate in a second DNA segment. This strand passage mechanism generates a requisite covalent enzyme-cleaved DNA intermediate, known as the cleavage complex. Under normal circumstances, the cleavage complex exists in a rapid cleavage/religation reaction equilibrium. However, when DNA tracking systems attempt to traverse topoisomerase-cleaved DNA complexes, the double-stranded DNA breaks cannot be rejoined by the enzyme and require DNA recombination/repair mechanisms to reestablish the integrity of the genetic material. In sufficient numbers, these strand breaks can overwhelm the repair processes and result in genotoxic effects including chromosomal translocations and cell death. Chemicals that stabilize cleavage complexes and increase genome fragmentation by converting type II topoisomerases into DNA-damaging agents and are known as topoisomerase poisons. Previous work has shown that levels of DNA cleavage mediated by type II topoisomerase are differentially affected by the handedness of DNA supercoil geometry. Human topoisomerase IIɑ and topoisomerase IIβ maintain higher levels of DNA cleavage complexes with negatively-supercoiled [(-)SC] DNA than with positively-supercoiled [(+)SC] substrates. However, it is not known whether increased DNA cleavage correlates with increased stability of cleavage complexes. We hypothesized that the stability of cleavage complexes may also vary by DNA supercoil geometry and enzyme type. To test our hypotheses, we assessed cleavage complex stability by measuring persistence of DNA cleavage in the presence or absence of human type II topoisomerase poisons. We determined that human topoisomerase IIɑ-mediated cleavage with (-)SC DNA persisted longer than with (+)SC DNA in the presence of etoposide and its derivative F14512, or amsacrine. The etoposide derivative TOP-53 generated a less pronounced difference in the persistence of topoisomerase IIɑ-mediated DNA cleavage. In contrast to the IIɑ isoform, the stability of IIβ-mediated DNA cleavage complexes across all poisons tested did not appear to vary by DNA supercoil handedness. We conclude that increased levels of DNA cleavage do not always correlate with greater stability of cleavage complexes. Both topoisomerases IIɑ and IIβ discriminate supercoil handedness during the DNA cleavage events, but how long enzyme-DNA cleavage complexes remain stable appears to be dependent on isoform. Although the IIɑ isoform both recognizes and prefers (-)SC over (+)SC DNA when maintaining cleavage complex stability, the IIβ isoform does not appear to discriminate. We are currently extending our experiments to bacteria with the Escherichia coli type II topoisomerases, gyrase and topoisomerase IV.