Topoisomerase II and etoposide — a tangled tale

Abstract

Etoposide, a chemotherapeutic poison of type IIA eukaryotic topoisomerases, promotes topoisomerase II to compact DNA by trapping DNA loops, creates DNA double-strand breaks, causes topo II to resist relocation, and pauses topisomerases’ ability to relax DNA supecoiling. Through these mechanisms, etoposide converts topoisomerase II into a roadblock to DNA processing.

The question

Etoposide is a broadly employed chemotherapeutic drug that poisons type IIA eukaryotic topoisomerases (topo IIs) — essential enzymes that relax DNA supercoiling via a strand-passage mechanism. Previous biochemical and structural studies show that etoposide stabilizes topo II-bound cleaved-DNA intermediates (known as DNA cleavage complexes), resulting in the conversion of a transient DNA break into a permanent one^1,2. Although etoposide is known to promote DNA breakage and induce cytotoxicity, how etoposide alters the dynamics and physical properties of topoisomerase action is unclear. Does etoposide change the ability of topo II to restructure the DNA? Can we directly detect the etoposide-stabilized cleavage complex? How does etoposide alter the ability of topo II to relocate on DNA? How does etoposide interfere with the ability of topo II to relax DNA supercoiling? The answers to these questions inform how etoposide ultimately induces cytotoxicity.

The discovery

We examined the action of etoposide on yeast topoisomerase II, human topoisomerase IIα, and human topoisomerase IIβ using three complementary single-molecule detection methods. First, optical tweezers DNA stretching for studying the overall DNA configurations, such as DNA compaction, DNA release, loop formation, and DNA breakage³. Second, optical tweezers DNA unzipping for high-resolution mapping of protein–DNA interactions and mimicking the motor removal of a bound protein⁴. Finally, magnetic tweezers DNA twisting for studies of supercoiling relaxation of topoisomerases⁵.

We found that etoposide enhances topo II’s ability to compact DNA by trapping DNA loops, and that loop trapping requires ATP hydrolysis, suggesting that loop trapping occurs after ATP hydrolysis but before strand ejection from the enzyme (Fig. 1a). We showed that our stretching and unzipping methods are highly sensitive for the detection of DNA tether breakage and, using these methods, we found that etoposide significantly enhances topo II-mediated DNA breaks only in the presence of hydrolyzable ATP (Fig. 1a). We also observed that topo II is mobile along DNA in the absence of etoposide but resists relocation and removal in the presence of etoposide (Fig. 1b). Interestingly, in addition to etoposide decreasing the stability of topo II dimers, it also increases the ability of the enzyme to act as a stable barrier and roadblock to DNA processing. Finally, we determined that etoposide induces prolonged pauses in topo II activity that interrupt the processive relaxation of supercoiled DNA, giving rise to a “burst-and-pause” behavior. These prolonged pauses correlate with the trapping of supercoiled loops.

Fig.1 |.

Etoposide promotes topo II-generated DNA breaks and stabilizes topo II-captured DNA loops. (a) Representative force extension curves of a DNA tether in the presence of yeast topo II, with and without ATP and etoposide. (b) Etoposide stabilizes the cleavage complex as well as topo II capture of a DNA crossover only in the presence of ATP hydrolysis

The implications

Etoposide has been used in chemotherapy since 1983 and is a valuable agent in treating a broad range of cancers. Despite its utility, a unified mechano-chemical view of its effect on topo II dynamics and action had been missing. Using highly sensitive single-molecule assays, we have revealed complex and sometimes unexpected behaviors.

Our finding that etoposide traps topoisomerase-mediated DNA loops could also mean that a topo II molecule bound to etoposide could lock two distant DNA segments from the same chromosome or two different chromosomes, significantly altering chromosome structure and topology. In addition, stably bound topoisomerase II has previously been proposed to interfere with replication and transcription activity in vivo. We now provide direct evidence to support this possibility. These experiments also highlight the importance of hydrolyzable ATP in enabling etoposide to promote topo II-mediated DNA loop trapping and DNA breakage.

This work demonstrates that single-molecule experiments can be a valuable, and often overlooked, addition to studies affecting human health. We anticipate that the techniques used here will be beneficial in studying a broad range of topo inhibitors and poisons, serving as sensitive screening tools for defining drug mechanisms and enzyme isoform specificity and providing valuable insights for end-user treatments.

EXPERT OPINION

“Le et al. use single-molecule assays to address how etoposide influences the topo II-DNA interaction enzymatic dynamics, concluding that etoposide stabilizes the double-stranded break status of topo II-DNA interaction and leads to a topological lock of both the G-segment and T-segment. The question the authors want to answer in this manuscript is significant given the broad application of etoposide in cancer therapy and poor understand of the mechanism of etoposide.”

Ahmet Yildiz, University of California Berkeley, Berkeley, CA, USA.

BEHIND THE PAPER

The most exciting moment was when we observed that etoposide promotes topo II trapping of DNA loops, and that enhanced trapping only occurs in the presence of hydrolyzable ATP. This finding suggests that etoposide stabilizes a state where the T-segment is likely trapped between the DNA gate and a closed C-gate of topo II after strand passage upon ATP hydrolysis. We were excited about this discovery because our single-molecule data seem to have captured a new and important consequence of etoposide-binding that has been overlooked in the field. This discovery was also a nice demonstration of a synergistic collaboration that brings together expertise from our lab on single-molecule studies and from James Berger’s lab on biochemical and structural studies. T.T.L and M.D.W.

FROM THE EDITOR

"This work by Le et al stood out to me because it uses various single molecule methods to study how the chemotherapeutic agent etoposide interacts with its target topoisomerase II proteins leading to double strand break of DNA. The findings not only reveal the detailed mechanism of action of etoposide, but also provide insight into DNA repair mechanism mediated by topoisomerase II proteins." Editorial Team, Nature Chemical Biology.