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. 2006 Mar 7;45(9):3040-50.
doi: 10.1021/bi051987q.

The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs

A Kathleen McClendon  1 , Neil Osheroff
Affiliations

The geometry of DNA supercoils modulates topoisomerase-mediated DNA cleavage and enzyme response to anticancer drugs

A Kathleen McClendon et al. Biochemistry. .

Abstract

Collisions with DNA tracking systems are critical for the conversion of transient topoisomerase-DNA cleavage complexes to permanent strand breaks. Since DNA is overwound ahead of tracking systems, cleavage complexes most likely to produce permanent strand breaks should be formed between topoisomerases and positively supercoiled molecules. Therefore, the ability of human topoisomerase IIalpha and IIbeta and topoisomerase I to cleave positively supercoiled DNA was assessed in the absence or presence of anticancer drugs. Topoisomerase IIalpha and IIbeta maintained approximately 4-fold lower levels of cleavage complexes with positively rather than negatively supercoiled DNA. Topoisomerase IIalpha also displayed lower levels of cleavage with overwound substrates in the presence of nonintercalative drugs. Decreased drug efficacy was due primarily to a drop in baseline (i.e., nondrug) cleavage, rather than an altered interaction with the enzyme-DNA complex. Similar results were seen for topoisomerase IIbeta, but the effects of DNA geometry on drug-induced scission were somewhat less pronounced. With both topoisomerase IIalpha and IIbeta, intercalative drugs displayed greater relative cleavage enhancement with positively supercoiled DNA. This appeared to result from negative effects of high concentrations of intercalative agents on underwound DNA. In contrast to the type II enzymes, topoisomerase I maintained approximately 3-fold higher levels of cleavage complexes with positively supercoiled substrates and displayed an even more dramatic increase in the presence of camptothecin. These findings suggest that the geometry of DNA supercoils has a profound influence on topoisomerase-mediated DNA scission and that topoisomerase I may be an intrinsically more lethal target for anticancer drugs than either topoisomerase IIalpha or IIbeta.

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Figures

FIGURE 1
FIGURE 1
Human topoisomerase IIα maintains lower levels of DNA cleavage complexes with positively supercoiled plasmids in the absence or presence of etoposide. The ability of topoisomerase IIα to cleave negatively [(-)SC, open circles] or positively [(+)SC, closed circles] supercoiled pBR322 plasmid DNA in the presence of 0-100 μM etoposide is shown (left panel). Assays employed Mg2+ as the divalent cation. Error bars represent the standard deviation of four independent assays. The inset shows a representative ethidium bromide-stained agarose gel of DNA cleavage assays with negatively and positively supercoiled plasmids in the absence of topoisomerase IIα (-TII) and in the absence (no drug, ND) or presence (Etop) of etoposide. Linear DNA standards (Lin) also are shown. The positions of supercoiled (form I, FI), nicked circular (form II, FII), and linear molecules (form III, FIII) are indicated. DNA sites cleaved by topoisomerase IIα were mapped in negatively or positively supercoiled plasmids (right panel). Products of DNA cleavage assays were linearized and singly-end labeled with [32P]phosphate. The autoradiogram is representative of three independent assays. Reactions were carried out in the presence of 0 or 100 μM etoposide, and utilized either Mg2+ or Ca2+ as the divalent cation. DNA from reactions that lacked topoisomerase IIα (-TII) are shown.
FIGURE 2
FIGURE 2
Etoposide inhibits the ligation of negatively or positively supercoiled DNA by human topoisomerase IIα. A time course of ligation in the presence of negatively [(-)SC, open symbols] or positively [(+)SC, closed symbols] supercoiled pBR322 plasmid is shown. DNA ligation was monitored in the absence (boxes) or presence (circles) of 100 μM etoposide (+Etop). The initial level of DNA cleavage was set to 100% and the rate of ligation was determined by quantifying the loss of the cleaved DNA over time. Error bars represent the standard deviation of three independent assays.
FIGURE 3
FIGURE 3
Effects of DNA superhelical geometry on drug-induced DNA cleavage mediated by human topoisomerase IIα. The ability of topoisomerase IIα to cleave negatively [(-)SC DNA, open bars] or positively [(+)SC DNA, closed bars] supercoiled pBR322 plasmid DNA in the presence of various topoisomerase II-targeted drugs is shown. All drugs were used at a concentration of 50 μM, except for CP-115,953, which was used at 5 μM (multiple DNA cleavage events per plasmid were observed at higher concentrations of CP-115,953). Error bars represent the standard deviation of 3 or 4 independent assays.
FIGURE 4
FIGURE 4
Effects of ethidium bromide intercalation on DNA cleavage mediated by human topoisomerase IIα. The ability of topoisomerase IIα to cleave negatively [(-)SC, open circles] or positively [(+)SC, closed circles] supercoiled pBR322 plasmid DNA in the presence of 0-25 μM ethidium bromide is shown (left panel). Error bars represent the standard deviation of three independent assays. The inset shows a representative gel of DNA intercalation assays using negatively supercoiled plasmids in the absence of topoisomerase IIα (-TII), or in the absence (0 μM) or presence (1-25 μM) of ethidium bromide (see Experimental Procedures for the interpretation of intercalation assays). The positions of supercoiled (FI) and nicked circular (FII) molecules are indicated. Relative DNA cleavage of linear plasmid, or negatively or positively supercoiled molecules in the absence (-EtBr, open bars) or presence (+EtBr, closed bars) of 10 μM eithdium bromide also is depicted (right panel). Relative DNA cleavage was calculated by normalizing levels of scission in the absence of ethidium bromide to a value of 1.0. Error bars represent the standard deviation of three independent assays for supercoiled substrates, or the standard error of the mean for two independent assays for linear DNA.
FIGURE 5
FIGURE 5
Effects of amsacrine and TAS-103 intercalation on DNA cleavage mediated by human topoisomerase IIα. The ability of topoisomerase IIα to cleave negatively [(-)SC, open circles] or positively [(+)SC, closed circles] supercoiled pBR322 plasmid DNA in the presence of 0-500 μM amsacrine (left panel) or 0-200 μM TAS-103 (right panel) is shown. Error bars represent the standard deviation of three independent assays. The insets show representative DNA intercalation assays in the absence of topoisomerase IIα (-TII), or in the absence (0 μM) or presence of the respective drug (see Experimental Procedures for the interpretation of intercalation assays). The positions of supercoiled (FI) and nicked circular (FII) molecules are indicated.
FIGURE 6
FIGURE 6
Human topoisomerase IIβ maintains lower levels of DNA cleavage complexes with positively supercoiled plasmids in the absence or presence of etoposide. The ability of topoisomerase IIβ to cleave negatively [(-)SC, open circles] or positively [(+)SC, closed circles] supercoiled pBR322 plasmid DNA is shown (left panel). Error bars represent the standard deviation of four independent assays. DNA cleavage in the presence of 0-100 μM etoposide also is depicted (right panel). Error bars represent the standard deviation of three independent assays. The inset shows a representative ethidium bromide-stained agarose gel of DNA cleavage assays with negatively and positively supercoiled plasmids in the absence of topoisomerase IIβ (-TII) and in the absence (no drug, ND) or presence (Etop) of etoposide. Linear DNA standards (Lin) also are shown. The positions of supercoiled (FI), nicked circular (FII), and linear molecules (FIII) are indicated.
FIGURE 7
FIGURE 7
Effects of DNA superhelical geometry on drug-induced DNA cleavage mediated by human topoisomerase IIβ. The ability of topoisomerase IIβ to cleave negatively [(-)SC DNA, open bars] or positively [(+)SC DNA, closed bars] supercoiled pBR322 plasmid DNA in the presence of various topoisomerase II-targeted drugs is shown. All drugs were used at a concentration of 50 μM, except for CP-115,953, which was used at 5 μM (multiple DNA cleavage events per plasmid were observed at higher concentrations of CP-115,953). Error bars represent the standard deviation of 3 or 4 independent assays.
FIGURE 8
FIGURE 8
Human topoisomerase I maintains higher levels of DNA cleavage complexes with positively supercoiled plasmids in the absence or presence of camptothecin. The ability of topoisomerase I to cleave negatively [(-)SC, open circles] or positively [(+)SC, closed circles] supercoiled pBR322 plasmid DNA in the presence of 0-25 μM campthothecin is shown. Error bars represent the standard deviation of three independent assays. The inset shows a representative ethidium bromide-stained agarose gel of DNA cleavage assays with negatively and positively supercoiled plasmids in the absence of topoisomerase I (-TI) and in the absence (no drug, ND) or presence (Cpt) of 5 μM camptothecin. Linear DNA standards (Lin) also are shown. The positions of supercoiled (FI), nicked circular (FII), and linear molecules (FIII) are indicated. The inset also shows a bar graph highlighting topoisomerase I-mediated DNA cleavage of negatively (open bar) and positively (closed bar) supercoiled DNA in the absence of drug.
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