Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the opening of the double helix. This enzyme helps to regulate DNA under- and overwinding and removes knots and tangles from the genetic material. In order to carry out its critical physiological functions, topoisomerase II generates transient double-stranded breaks in DNA. Consequently, while necessary for cell survival, the enzyme also has the capacity to fragment the genome. The DNA cleavage/ligation reaction of topoisomerase II is the target for some of the most successful anticancer drugs currently in clinical use. However, this same reaction also is believed to trigger chromosomal translocations that are associated with specific types of leukemia. This article will familiarize the reader with the DNA cleavage/ligation reaction of topoisomerase II and other aspects of its catalytic cycle. In addition, it will discuss the interaction of the enzyme with anticancer drugs and the mechanisms by which these agents increase levels of topoisomerase II-generated DNA strand breaks. Finally, it will describe dietary and environmental agents that enhance DNA cleavage mediated by the enzyme.

Type II topoisomerases are required for the management of DNA tangles and supercoils1, and are targets of clinical antibiotics and anti-cancer agents2. These enzymes catalyze the ATP-dependent passage of one DNA duplex (the transport or T-segment) through a transient, double-stranded break in another (the gate or G-segment), navigating DNA through the protein using a set of dissociable internal interfaces, or “gates”3,4. For more than 20 years, it has been established that a pair of dimer-related tyrosines, together with divalent cations, catalyze G-segment cleavage5–7. Recent efforts have proposed that strand scission relies on a “two-metal mechanism”8–10, a ubiquitous biochemical strategy that supports vital cellular processes ranging from DNA synthesis to RNA self-splicing11,12. Here we present the structure of the DNA-binding and cleavage core of Saccharomyces cerevisiae topo II covalently linked to DNA through its active-site tyrosine at 2.5 Å resolution, revealing for the first time the organization of a cleavage-competent type II topoisomerase configuration. Unexpectedly, metal-soaking experiments indicate that cleavage is catalyzed by a novel variation of the classic two-metal approach. Comparative analyses extend this scheme to explain how distantly-related type IA topoisomerases cleave single-stranded DNA, unifying the cleavage mechanisms for these two essential enzyme families. The structure also highlights a hitherto undiscovered allosteric relay that actuates a molecular “trapdoor” to prevent subunit dissociation during cleavage. This connection illustrates how an indispensable chromosome-disentangling machine auto-regulates DNA-breakage to prevent the aberrant formation of mutagenic and cytotoxic genomic lesions.

DNA is essentially an extremely long double-stranded rope in which the two strands are wound about one another. As a result, topological properties of the genetic material, including DNA underwinding and overwinding, knotting, and tangling profoundly influence virtually every major nucleic acid process. Despite the importance of DNA topology, it is a conceptionally difficult subject to teach because it requires students to visualize three-dimensional relationships. This article will familiarize the reader with the concept of DNA topology and offer practical approaches and demonstrations to teaching this ''knotty'' subject in the classroom. Furthermore, it will discuss topoisomerases, the enzymes that regulate the topological state of DNA in the cell. These ubiquitous enzymes perform a number of critical cellular functions by generating transient breaks in the double helix. During this catalytic event, topoisomerases maintain genomic stability by forming covalent phosphotyrosyl bonds between active site residues and the newly generated DNA termini. Topoisomerases are essential for cell survival. However, because they cleave the genetic material, these enzymes also have the potential to fragment the genome. This latter feature of topoisomerases is exploited by some of the most widely prescribed anticancer and antibacterial drugs currently in clinical use. Finally, in addition to curing cancer, topoisomerase action also has been linked to the induction of specific types of leukemia.Keywords: nucleic acid structure, function, and processing, nucleic acid enzymology, molecular biology, medical biochemistry.Although the genetic information encoded in DNA is embodied in a one-dimensional array of bases, it is the three-dimensional structure of the genetic material that controls how this information is replicated, expressed, and recombined in the cell [1][2][3][4][5]. Because DNA is double-stranded and compressed into a crowded cellular environment, some of the most important three-dimensional relationships in the double helix are topological in nature.How is the concept of topology applied to the genetic material? As long as the ends of DNA are fixed in space, topological relationships are defined as those that can be altered only by breaking one or both strands of the double helix. For all practical purposes, we always can consider the ends of cellular DNA to be anchored and unable to rotate [3][4][5][6]. This is because there is a high frictional energy associated with the extreme length of chromosomes, plasmids and bacterial chromosomes often are circular (and therefore have no ends), and chromosomal DNA is tethered to membranes in bacteria and to the chromosome scaffold in eukaryotes [3][4][5][6]. Topological relationships in DNA include underwinding and overwinding, knotting, and tangling.DNA topology should be an integral component of biochemistry and molecular biology curricula for a number of reasons, including:

The DNA cleavage reaction of human topoisomerase IIα is critical to all of the physiological and pharmacological functions of the protein. While it has long been known that the type II enzyme requires a divalent metal ion in order to cleave DNA, the role of the cation in this process is not known. To resolve this fundamental issue, the present study utilized a series of divalent metal ions with varying thiophilicities in conjunction with DNA cleavage substrates that replaced the 3′-bridging oxygen of the scissile bond with a sulfur atom (i.e. 3′-bridging phosphorothiolates). Rates and levels of DNA scission were greatly enhanced when thiophilic metal ions were included in reactions that utilized sulfur-containing substrates. Based on these results and those of reactions that employed divalent cation mixtures, we propose that topoisomerase IIα mediates DNA cleavage via a two-metal-ion mechanism. In this model, one of the metal ions makes a critical interaction with the 3′-bridging atom of the scissile phosphate. This interaction greatly accelerates rates of enzyme-mediated DNA cleavage, and most likely is needed to stabilize the leaving 3′-oxygen.

Etoposide is a topoisomerase II poison that is used to treat a variety of human cancers. Unfortunately, 2–3% of patients treated with etoposide develop treatment-related leukemias characterized by 11q23 chromosomal rearrangements. The molecular basis for etoposide-induced leukemogenesis is not understood, but is associated with enzyme-mediated DNA cleavage. Etoposide is metabolized by CYP3A4 to etoposide catechol, which can be further oxidized to etoposide quinone. A CYP3A4 variant is associated with a lower risk of etoposide-related leukemias, suggesting that etoposide metabolites may be involved in leukemogenesis. Although etoposide acts at the enzyme-DNA interface, several quinones poison topoisomerase II via redox-dependent protein adduction. The effects of etoposide quinone on topoisomerase IIα-mediated DNA cleavage have been examined previously. Although findings suggest that the activity of the quinone is slightly greater than that of etoposide, these studies were carried out in the presence of significant levels of reducing agents (which should reduce etoposide quinone to the catechol). Therefore, we examined the ability of etoposide quinone to poison human topoisomerase IIα in the absence of reducing agents. Under these conditions, etoposide quinone was ~5–fold more active than etoposide at inducing enzyme-mediated DNA cleavage. Consistent with other redox-dependent poisons, etoposide quinone inactivated topoisomerase IIα when incubated with the protein prior to DNA and lost activity in the presence of dithiothreitol. Unlike etoposide, the quinone metabolite did not require ATP for maximal activity and induced a high ratio of double-stranded DNA breaks. Our results support the hypothesis that etoposide quinone contributes to etoposide-related leukemogenesis.