Session: Current Topics in Biological Chemistry:

1,2-naphthoquinone, a cytotoxic and genotoxic metabolite of naphthalene, is an environmental pollutant found in diesel exhaust particles. Many quinones have been shown to act as topoisomerase II poisons, which are compounds that stabilize a covalent enzyme-cleaved DNA complex (cleavage complex) formed as part of the catalytic cycle of type II enzymes. The actions of topoisomerase II poisons generate genome-wide double-stranded breaks in the genetic material that have to be repaired by DNA recombination pathways. Due to its potential as a topoisomerase poison, the effects of 1,2-naphthoquinone on DNA cleavage mediated by human topoisomerase IIα and IIβ were examined. 1,2-naphthoquinone induced ~25% and ~14% double-stranded DNA breaks generated by human topoisomerase IIα and IIβ, respectively. Due to the higher efficacy of the compound against topoisomerase IIα over IIβ, the α isoform was used for additional mechanistic studies. 1,2-naphthoquinone induced covalent topoisomerase IIα-DNA cleavage complexes that were stable for at least 2 hours with no observed decrease in DNA scission. Additionally, the compound was a partial inhibitor of enzyme-mediated DNA ligation. Topoisomerase II poisons can be classified as interfacial or covalent. Interfacial poisons interact non-covalently at the enzyme-DNA interface and increase DNA cleavage by blocking ligation. Conversely, covalent poisons adduct the protein at residues outside of the active site and increase DNA cleavage by closing the N-terminal gate of topoisomerase II. Therefore, a series of experiments was performed to determine the mechanistic basis for the actions of 1,2-naphthoquinone. In contrast to results with etoposide (an interfacial poison), the activity of 1,2-naphthoquinone against topoisomerase IIα was abrogated in the presence of sulfhydryl and reducing agents. Additionally, the compound rapidly inhibited enzyme-mediated DNA scission (t_1/2 ~1.5 min) when incubated with topoisomerase IIα prior to the addition of DNA. Finally, 1,2-naphthoquinone induced virtually no cleavage with the catalytic core of topoisomerase IIα (which lacks the N-terminal gate). These results are all hallmarks of covalent poisons and strongly suggest that 1,2-naphthoquinone increases topoisomerase II-mediated double-stranded DNA scission by acting as a covalent poison of the type II enzymes. The effects of the compound on type II topoisomerases may explain some of the toxicity associated with 1,2-naphthoquinone in humans.

Neil Osheroff

Appearances