Quinone-Induced Enhancement of DNA Cleavage by Human Topoisomerase IIα: Adduction of Cysteine Residues 392 and 405†

Topoisomerase IIα is an essential enzyme that plays important roles in a number of growth related processes in human cells, including DNA replication and chromosome segregation. The enzyme removes superhelical twists, knots, and tangles from the genetic material by generating transient double-stranded breaks in the backbone of DNA (1-7). To maintain genomic integrity during this cleavage event, the enzyme forms covalent bonds between active site tyrosyl residues and the 5’-DNA termini created by scission of the double helix (8-10). Although these transient covalent topoisomerase IIα-cleaved DNA complexes (i.e., cleavage complexes) are requisite intermediates in the catalytic cycle of the enzyme, their presence is potentially deleterious to the cell. When a DNA tracking system, such as a replication fork, attempts to traverse a cleavage complex, the transient enzyme-bound DNA break often is converted to a permanent strand break (1-4, 7). Thus, conditions that significantly increase the concentration of topoisomerase IIα-DNA cleavage complexes trigger illegitimate recombination, chromosomal aberrations, and cell death pathways (4, 11-16). Agents that increase levels of topoisomerase IIα-mediated DNA strand breaks are termed topoisomerase II poisons to distinguish their mechanism of action from compounds that act by inhibiting the overall catalytic activity of the enzyme (i.e., catalytic inhibitors) (4, 15, 17-19). Topoisomerase II poisons play important roles in both the treatment and the development of human cancers. Approximately one-half of all cancer chemotherapy regimens contain drugs that target the type II enzyme. Furthermore, several topoisomerase II poisons that are naturally found in fruits and vegetables, such as bioflavonoids, are believed to have chemopreventative properties (20-23). Conversely, a small number of patients who receive therapy that includes topoisomerase II-targeted drugs develop secondary leukemias. These drug-related leukemias commonly feature aberrations (deletions, duplications, and translocations) in the mixed-lineage leukemia (MLL) gene at chromosomal band 11q23 (13, 14, 24-29). In addition, the same bioflavonoids that are beneficial for adult health, when ingested during pregnancy, are believed to increase the risk of infant leukemias that include MLL rearrangements (30-34). Recently, a number of toxic quinone-based metabolites of drugs or environmental pollutants have been found to be potent human topoisomerase II poisons (35). These include N-acetyl-p-benzoquinone imine (derived from acetaminophen) (36), benzoquinone (derived from benzene) (37, 38), as well as several polychlorinated biphenyl (PCB)1 metabolites (39). Exposure to these compounds causes DNA strand breaks and other chromosomal aberrations, and has been linked to a variety of human health problems, including cancer (40-44). To this point, the accumulation of benzoquinone in the bone marrow is believed to contribute to the leukemogenic properties of benzene (41). Moreover, genetic deficiencies in NAD(P)H:quinone oxidoreductase 1, the enzyme that reduces benzoquinone to the less reactive hydroquinone, are linked to the formation of leukemias with MLL rearrangements (45-49). The quinone-based topoisomerase II poisons differ from “classic” topoisomerase II poisons (such as etoposide and bioflavonoids) in two ways. First, quinones are mixed-function inhibitors of the type II enzyme: they increase DNA scission when added to a topoisomerase II-DNA complex, but inhibit scission when incubated with the enzyme prior to the addition of nucleic acids (36-39, 50-54). Second, in contrast to compounds like etoposide (which interact with topoisomerase II in a non-covalent manner), quinones act by covalently attaching to the protein (35-39, 53). Because of the human health concerns of quinone-based topoisomerase II poisons and their unusual mode of action against the type II enzyme, sites of quinone adduction on human topoisomerase IIα were mapped by mass spectrometry. Four cysteine residues were identified and mutated to alanine residues. Two of the mutant enzymes, top2αC392A and top2αC405A, displayed decreased sensitivity to benzoquinone and the PCB quinone 4’Cl-2,5pQ. These findings strongly suggest that adduction of cysteine residues 392 and 405 play a role in the actions of quinones against human topoisomerase IIα.