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. 2011 Feb;39(3):1014-22.
doi: 10.1093/nar/gkq822. Epub 2010 Sep 19.

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Amanda C Gentry  1 , Sissel Juul, Christopher Veigaard, Birgitta R Knudsen, Neil Osheroff
Affiliations

The geometry of DNA supercoils modulates the DNA cleavage activity of human topoisomerase I

Amanda C Gentry et al. Nucleic Acids Res. 2011 Feb.

Abstract

Human topoisomerase I plays an important role in removing positive DNA supercoils that accumulate ahead of replication forks. It also is the target for camptothecin-based anticancer drugs that act by increasing levels of topoisomerase I-mediated DNA scission. Evidence suggests that cleavage events most likely to generate permanent genomic damage are those that occur ahead of DNA tracking systems. Therefore, it is important to characterize the ability of topoisomerase I to cleave positively supercoiled DNA. Results confirm that the human enzyme maintains higher levels of cleavage with positively as opposed to negatively supercoiled substrates in the absence or presence of anticancer drugs. Enhanced drug efficacy on positively supercoiled DNA is due primarily to an increase in baseline levels of cleavage. Sites of topoisomerase I-mediated DNA cleavage do not appear to be affected by supercoil geometry. However, rates of ligation are slower with positively supercoiled substrates. Finally, intercalators enhance topoisomerase I-mediated cleavage of negatively supercoiled substrates but not positively supercoiled or linear DNA. We suggest that these compounds act by altering the perceived topological state of the double helix, making underwound DNA appear to be overwound to the enzyme, and propose that these compounds be referred to as 'topological poisons of topoisomerase I'.

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Figures

Figure 1.
Figure 1.
Topoisomerase I maintains higher levels of cleavage complexes with positively supercoiled DNA. The ability of increasing concentrations of human topoisomerase I to cleave positively [(+)SC, closed circles] and negatively [(−)SC, open circles] supercoiled pBR322 plasmid DNA is shown. Error bars represent the standard deviation of at least three independent experiments.
Figure 2.
Figure 2.
Effects of DNA supercoil handedness on topoisomerase I-mediated DNA cleavage in the presence of anticancer drugs. The ability of topoisomerase I to cleave positively [(+)SC, closed circles] and negatively [(−)SC, open circles] supercoiled pBR322 plasmid DNA in the presence of 0–10 µM camptothecin (A) or topotecan (B) is shown. Error bars represent the standard deviation of four independent experiments.
Figure 3.
Figure 3.
Effects of DNA supercoil handedness on sites of topoisomerase I-mediated DNA cleavage. DNA sites cleaved by topoisomerase I were mapped in negatively [(−)SC] and positively [(+)SC] supercoiled pBR322 plasmid DNA in the absence (TOP1) or presence (TPT) of 5 µM topotecan. Untreated DNA is shown as a control (DNA). Following cleavage assays, plasmids were linearized and singly-end-labeled with [32P]-phosphate as described under ‘Materials and Methods’ section. The autoradiogram is representative of three independent experiments. Size markers that were 300 bp and smaller were derived from DNA sequence ladders and those that were 350 bp and larger were derived from restriction digests.
Figure 4.
Figure 4.
Effects of DNA supercoil handedness on topoisomerase I-mediated DNA ligation in the presence of anticancer drugs. The ability of topoisomerase I to ligate positively [(+)SC, closed circles] and negatively [(−)SC, open circles] supercoiled pBR322 plasmid DNA was monitored in the presence of 10 µM camptothecin (A) or 5 µM topotecan (B). DNA ligation was initiated by the addition of 300 mM NaCl. Levels of cleavage at time zero were set to 100%. Error bars represent the standard deviation of three independent experiments.
Figure 5.
Figure 5.
Effects of DNA intercalators on topoisomerase I-mediated cleavage of plasmid DNA. Cleavage of positively [(+)SC, closed circles] and negatively [(−)SC, open circles] supercoiled pBR322 plasmid DNA was monitored in the presence of ethidium bromide (EtBr), TAS-103, 9-aminoacridine (9-AA) or amsacrine (m-AMSA). Data were plotted as relative (i.e. fold) DNA cleavage enhancement for simplicity and to aid in visualizing the effects of intercalators on topoisomerase I-mediated cleavage of positively and negatively supercoiled substrates. Fold DNA cleavage enhancement was calculated by normalizing levels of scission in the absence of intercalator to a relative value of 1.0. Error bars represent the standard deviation of at least three independent experiments. Insets show representative gels of DNA intercalation assays using relaxed plasmids in the absence of enzyme (DNA) or in the presence of the indicated concentration (µM) of compound (see ‘Materials and Methods’ section for the interpretation of intercalation assays). Note that intercalation assays are designed to monitor the DNA relaxation activity of topoisomerase I. Consequently, reactions are terminated under conditions that do not trap enzyme–DNA cleavage complexes. The positions of supercoiled (FI) and nicked circular (FII) molecules are indicated.
Figure 6.
Figure 6.
Effects of anticancer drugs and DNA intercalators on topoisomerase I-mediated cleavage of linear DNA. The ability of anticancer drugs [5 µM camptothecin (Cpt) or 10 µM topotecan (Tpt)] and DNA intercalating agents [20 µM ethidium bromide (EtBr), 20 µM TAS-103 (TAS), 100 µM 9-aminoacridine (9-AA) or 200 µM amsacrine (AMSA)] to enhance cleavage of a 3′-end labeled DNA substrate was determined. The autoradiograph is representative of three independent experiments. Size markers are described in Figure 3.
Figure 7.
Figure 7.
Effects of DNA intercalators on topoisomerase I-mediated cleavage of plasmid DNA in the presence of anticancer drugs. Cleavage of positively [(+)SC, closed circles] and negatively [(−)SC, open circles] supercoiled pBR322 plasmid DNA was monitored in the presence of 0–20 µM ethidium bromide and either 2.5 µM camptothecin (A) or 5 µM topotecan (B). Error bars represent the standard deviation of at least three independent experiments.
Figure 8.
Figure 8.
Effects of ethidium bromide on topoisomerase I-mediated DNA cleavage in cultured human CEM cells. Cells were treated for 1 h with no drug (ND), 10 µM ethidium bromide (EtBr), 5 µM topotecan (Tpt) or both ethidium bromide and topotecan (Tpt + EtBr). Topoisomerase I-DNA complexes were monitored using the ICE bioassay (see inset for a representative blot). Levels of topoisomerase I-mediated DNA cleavage in the absence of drug were set to a relative value of 1. Error bars represent the standard deviation of six independent experiments.
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