Type II topoisomerases are enzymes that regulate DNA supercoiling. Humans encode two type II topoisomerase isoforms, IIα and IIβ. Type II topoisomerases function by passing an intact double helix through a transient double-stranded break in a second DNA segment, generating a requisite covalent enzyme-cleaved DNA intermediate known as the cleavage complex. When DNA tracking systems attempt to traverse topoisomerase-cleaved DNA complexes, double-stranded DNA breaks cannot be rejoined by the enzyme and require DNA recombination/repair mechanisms to reestablish genomic integrity. In sufficient numbers, these DNA breaks can overwhelm repair processes, resulting in genotoxic effects such as chromosomal translocations and cell death. Chemicals that stabilize cleavage complexes convert type II topoisomerases into DNA-damaging agents and are termed topoisomerase poisons. DNA supercoil handedness affects levels of DNA cleavage mediated by type II topoisomerases. Human topoisomerases IIα and IIβ maintain higher DNA cleavage with negatively-supercoiled [(-)SC] than positively-supercoiled [(+)SC] DNA. However, it is unclear if increased DNA cleavage correlates with increased cleavage complex stability. We hypothesized that cleavage complex stability may also vary by DNA supercoil handedness and enzyme type. To test our hypotheses, we assessed cleavage complex stability by measuring the persistence of DNA cleavage in the presence or absence of topoisomerase II poisons. Topoisomerase IIα-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 similar but less pronounced difference in the persistence of topoisomerase IIα-mediated DNA cleavage. In contrast to the IIα isoform, the stability of IIβ-mediated DNA cleavage complexes formed with poisons did not vary by supercoil handedness. We conclude that increased cleavage levels do not always correlate with greater cleavage complex stability. Both topoisomerases IIα and IIβ discriminate supercoil handedness during the DNA cleavage events, but the stability of cleavage complexes appears to be isoform dependent. Although the IIα isoform 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 <i>Escherichia coli </i>type II topoisomerases, gyrase and topoisomerase IV.

DNA supercoil handedness differentially affects the stability of DNA cleavage complexes formed by different type II topoisomerases

ACS Spring Meeting 2021

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