Quinone-induced enhancement of DNA cleavage by human topoisomerase IIalpha: adduction of cysteine residues 392 and 405

Abstract

Several quinone-based metabolites of drugs and environmental toxins are potent topoisomerase II poisons. These compounds act by adducting the protein and appear to increase levels of enzyme-DNA cleavage complexes by at least two potentially independent mechanisms. Treatment of topoisomerase IIalpha with quinones inhibits DNA religation and blocks the N-terminal gate of the protein by cross-linking its two protomer subunits. It is not known whether these two effects result from adduction of quinone to the same amino acid residue(s) in topoisomerase IIalpha or whether they are mediated by modification of separate residues. Therefore, this study identified amino acid residues in human topoisomerase IIalpha that are modified by quinones and determined their role in the actions of these compounds as topoisomerase II poisons. Four cysteine residues were identified by mass spectrometry as sites of quinone adduction: Cys170, Cys392, Cys405, and Cys455. Mutations (Cys --> Ala) were individually generated at each position. Only mutations at Cys392 or Cys405 reduced sensitivity ( approximately 50% resistance) to benzoquinone. Top2alphaC392A and top2alphaC405A displayed faster rates ( approximately 2-fold) of DNA religation than wild-type topoisomerase IIalpha in the presence of the quinone. In contrast, as determined by DNA binding, protein clamp closing, and protomer cross-linking experiments, mutations at Cys392 and Cys405 did not affect the ability of benzoquinone to block the N-terminal gate of topoisomerase IIalpha. These findings indicate that adduction of Cys392 and Cys405 is important for the actions of quinones against the enzyme and increases levels of cleavage complexes primarily by inhibiting DNA religation.

Figure 1

DNA cleavage enhancement by human topoisomerase IIα in the presence of plumbagin. Upper Right: The structures of plumbagin and benzoquinone are shown. Reactive sites on the compounds are indicated by stars. Left: An autoradiogram of a polyacrylamide gel of DNA cleavage reactions is shown. Assays contained no compound (None), 12.5 μM etoposide, or 25 μM benzoquinone or 100–500 μM plumbagin. A DNA control is shown in the far left lane. Data are representative of two independent experiments. Lower Right: Results of topoisomerase II protein clamp closing assays are shown. The bar graph represents levels of salt-stable bound DNA formed when topoisomerase II-DNA complexes were treated with no drug (None), 250 μM plumbagin or 100 μM benzoquinone. Error bars represent the standard deviation of four independent experiments.

Figure 2

Plumbagin adducts to cysteine residues in human topoisomerase IIα. Residues adducted by quinones were identified by mass spectrometry. A composite of the crystal structures of the yeast catalytic core and N-terminal domain is shown and the locations of the homologous cysteine residues adducted in human topoisomerase IIα are indicated by shaded circles. These residues were mutated to alanines using mutagenesis PCR to evaluate their role in quinone action. Adapted from Refs. (68, 69).

Figure 3

DNA cleavage activity of mutant human topoisomerase IIα enzymes. Panel A: Cleavage activity was assessed using 0–800 nM topoisomerase IIα in the absence of quinones. Assay mixtures contained wild-type enzyme (WT, open squares), top2αC170A (C170A, open circles), top2αC392A (C392A, filled squares), top2αC405A (C405A, filled circles), or top2αC392/405A (C392/405A, open triangles). Panel B: DNA cleavage reactions were carried out in the presence of 0–200 μM etoposide. Panel C: DNA cleavage reactions were carried out in the presence of 0–200 μM benzoquinone. Panel D: DNA cleavage reactions were carried out in the presence of 0–200 μM 4’Cl-2,5pQ. Error bars represent the standard deviation of at least three independent experiments.

Figure 4

Quinone-resistant mutant human topoisomerase IIα enzymes display higher rates of DNA religation in the presence of benzoquinone. Left Panel: DNA religation reactions were carried out in the presence of 100 μM benzoquinone. Assay mixtures contained wild-type enzyme (WT, open squares), top2αC170A (C170A, open circles), top2αC392A (C392A, filled squares), top2αC405A (C405A, filled circles), or top2αC392/405A (C392/405A, open triangles). Samples were incubated at 37 °C to establish DNA cleavage/religation equilibria. Reactions were shifted to 0 °C to initiate religation. DNA religation was quantified by the loss of linear cleaved molecules. Right Panel: Representative DNA religation data at 10 s is shown. DNA cleavage/religation equilibria were established in the presence (filled bars) or absence (None, open bars) of benzoquinone. Error bars represent the standard deviation of at least three independent experiments.

Figure 5

Substrate-dependent effects of benzoquinone on mutant topoisomerase IIα-DNA binding. Assays employed a negatively supercoiled circular plasmid (shaded bars), a linear plasmid (open bars), or a duplex 50-mer oligonucleotide (filled bars) as the substrate. Enzymes were incubated with 100 μM benzoquinone for 5 min prior to the addition of DNA. DNA binding in the absence of benzoquinone was set to 100%. Error bars represent the standard deviation of at least three independent experiments.

Figure 6

Benzoquinone blocks the N-terminal gate of mutant human topoisomerase IIα enzymes. Filter binding assays were used to analyze the salt-stable closed-clamp of topoisomerase II. Enzyme-DNA complexes were established and further incubated in the absence (None, open bars) or presence (filled bars) of 100 μM benzoquinone. Samples were applied to glass fiber filters, DNA was eluted by sequential washes in low salt, high salt, and SDS, and eluted samples were subjected to electrophoresis in an agarose gel. Salt-stable non-covalent enzyme-DNA complexes were quantified by the amount of plasmid that did not elute until the SDS wash relative to the total plasmid eluted in all three washes. Error bars represent the standard deviation of at least three independent experiments.

Figure 7

Benzoquinone crosslinks the protomer subunits of mutant human topoisomerase IIα enzymes. Assay mixtures contained 135 nM wild-type enzyme (WT, open squares), top2αC170A (C170A, open circles), top2αC392A (C392A, filled squares), top2αC405A (C405A, filled circles), or top2αC392/405A (C392/405A, open triangles) that was treated with 100 μM benzoquinone for 0–4 min. Samples were subject to electrophoresis in a polyacrylamide gel and protein was visualized by coomassie staining. The level of 170 kDa enzyme protomer that was present in the absence of benzoquinone was set to 1. Error bars represent the standard deviation of at least three independent experiments.