Deep within your molecular biology are the paradoxical molecules called “Topoisomerase II”, which are potentially very dangerous in that they continuously and literally splice up your entire genome––and yet no life on Earth could ever exist or function without them. They temporarily induce immense damage to all your DNA…and then repair it perfectly. How this molecule functions on a near-atomic scale remains mostly a mystery but ALL life on Earth requires this molecule to exist and function, as do cancer cells. And it once also seemed that the key to finally curing cancer was to shut down this molecule so it cannot perform its function on DNA within cancerous cells.

In order for you to exist from microsecond to microsecond your entire genome–the sum total of your DNA–must be continuously maintained and regulated and repaired. This is done through an arsernal of enzymes, most crucially Topoisomerase II. The function of this enzyme/protein is to perform a crucial mathematical function: to remove knots and tangles and relieve overwinding/torsional stresses (positive supercoiling)  within DNA. If DNA has knots or tangles, then tracking enzymes like RNA polymerase (responsible for protein transcription and renewing all your proteins) and DNA polymerase (which is initially responsible for DNA replication and thus cell division) could never function. They move along DNA like beads on a string and cannot proceed if they enounter knots or tangles.

However Topoisomerase II removes the DNA knots by performing a very dangerous “double-strand break” on your DNA.: Much like Alexander the Great’s solution to the Gordian Knot, they cut it open like a sword. The cut knot is ‘unknotted’ or removed and then the strand is resealed. However, it does more than that: it ensures DNA remains “underwound” or “negatively supercoiled” with a specific ‘Gaussian linking number’, (A formula first written down by Gauss in 1820.)). The DNA of all life on Earth shares this property.This molecule seems to know all the mathematical tricks from knot theory textbooks. However, at any instant, these molecules are splicing up your entire genome/DNA like scissors cutting string into billions of pieces…yet at the same time they reseal it all back together with all the knots removed…and so you continue to function and exist.

The class of cancer drugs called “Doxyrubicins” were designed to trap or ‘freeze’ Topoisomerase II so that the cancer DNA would remain knotted and tangled. Hence, it would not be able to replicate itself and the cancer cells would die. However, the cancer cells which survived the first rounds of treatment were able to unknot themselves with fewer Topoisomerase enzymes. Also, new genes can be turned on which enables the cells to expell the drug. Neverthleless, while not a cure, they have proven themselves to be effective drugs.”

……………………………………………………………………………………………………..(Follows closely: 738–748 Nucleic Acids Research, 2009, Vol. 37, No. 3) Topoisomerase II is an essential enzyme that is required for virtually every process that requires movement of DNA within the nucleus or the openingof 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.

A number of enzymes that catalyze essential physiological processes also have the capacity to damage the genome during the course of their normal activities. For example, while the cell requires DNA polymerases to copy the genetic material, these enzymes insert an incorrect base approximately every 107 nt (1). Consequently, in the absence of mismatch repair pathways, human DNA
polymerases would generate several hundred mutations every round of cell division. Furthermore, while DNA glycosylases initiate base-excision repair pathways, these enzymes can convert innocuous lesions to abasic sites
with far greater mutagenic potential (2). Finally, while cytochrome P450 enzymes play critical roles in detoxification pathways, they sometimes convert inert xenobiotic chemicals to compounds with mutagenic properties (3). Of all the enzymes required to sustain cellular growth, topoisomerase II is one of the most dangerous (4–8). As discussed below, this enzyme unwinds, unknots and
untangles the genetic material by generating transient double-stranded breaks in DNA (8–12). Although the cell cannot survive without topoisomerase II, the strand breaks that the enzyme generates have the potential to trigger cell death pathways or chromosomal translocations.

This article focuses on the mechanism by which topoisomerase
II cleaves the genetic material, the ability to exploit this reaction for the chemotherapeutic treatment of human cancers and the role of this reaction in triggering specific types of leukemia.
DNA TOPOLOGY
The existence of topoisomerases is necessitated by the structure of the double helix. Each human cell contains 2m of DNA that are compacted into a nucleus that is 10 mm in diameter (14,15). Because the genetic material is anchored to the chromosome scaffold and the two strands of the double helix are plectonemically coiled, accessing the genome is a complex topological challenge (11,12,16–18). Topological properties of DNA are those that can only be changed when the double helix is broken (12). Two aspects of DNA topology significantly affect nuclear processes. The first deals with topological relationships between the two strands of the double helix.

In all living systems, from bacteria to humans, DNA is globally underwound
(i.e. negatively supercoiled) by 6% (12,19–21). This is important because duplex DNA is merely the storage form for the genetic information. In order to
replicate or express, DNA must be separated. Since global underwinding of
the genome imparts increased single-stranded character to the double helix, negative supercoiling greatly facilitates strand separation (12,16–18). While negative supercoiling promotes many nucleic acid processes, DNA overwinding (i.e. positive supercoiling) inhibits them. The linear movement of tracking enzymes, such as helicases and polymerases, compresses the turns of the double helix into a shorter region (Figure 1) (12,19–21). Consequently, the double helix becomes increasingly overwound ahead of tracking systems. The positive supercoiling that results makes it more difficult to open the two strands of the double helix and ultimately blocks essential nucleic acid processes (10,12,16–18). The second aspect of DNA topology deals with relationships between separate DNA segments. Intramolecular knots (formed within the same DNA molecule) are
generated during recombination, and intermolecular tangles (formed between daughter DNA molecules) are produced during replication (Figure 1) (8,10,12,17).

DNA knots block essential nucleic acid processes because they make it impossible to separate the two strands of the double helix. Moreover, tangled DNA molecules cannot be segregated during mitosis or meiosis (8,10,12,17). Consequently, DNA knots and tangles can be lethal to cells if they are not resolved.
DNA TOPOISOMERASES
The topological state of the genetic material is regulated by enzymes known as topoisomerases (8,10,11,22,23). Topoisomerases are required for the survival of all organisms and alter DNA topology by generating transient breaks in the double helix (8,10,11,22,23). There are two major classes of topoisomerases, type I and type II, that are distinguished by the number of DNA strands that they cleave and the mechanism by which they alter the topological properties of the genetic material (8,10,11,22,23). Eukaryotic type I topoisomerases are monomeric
enzymes that require no high-energy cofactor (11,22,24). Type I enzymes are organized into two subclasses: type IA and type IB. These enzymes alter topology by creating transient single-stranded breaks in the DNA, followed by passage of the opposite intact strand through the break (type IA) or by controlled rotation of the helix around the break (type IB) (11,22,24). Type IA topoisomerases need divalent metal ions for DNA scission and attach covalently to the 50-terminal phosphate of the DNA (11,22,24). In contrast, type IB enzymes do not require divalent metal ions and covalently link to the 30-terminal phosphate (11,22,24).

As a result of their reaction mechanism, type I topoisomerases can modulate
DNA under- and overwinding, but cannot remove knots or tangles from duplex DNA. A number of excellent review articles on type I topoisomerases have
appeared recently (22,24,25). Consequently, these enzymes will not be discussed further in this article. Eukaryotic type II topoisomerases function as homodimers and require divalent metal ions and ATP for complete catalytic activity (5,8,26–28). These enzymes interconvert different topological forms of DNA by a ‘double-stranded DNA passage reaction’ that can be separated into a number of discrete steps (5,8,26–28). Briefly, type II topoisomerases (i) bind two separate segments of DNA, (ii) create a double-stranded break in one of the segments, (iii) translocate the second DNA segment through the cleaved nucleic acid ‘gate’, (iv) rejoin (i.e. ligate) the cleaved DNA, (v) release the translocated segment through a gate in the protein and (vi) close the protein gate and regain the ability to start a new round of catalysis (5,26–34). Because of their double-stranded DNA passage mechanism, type II topoisomerases can modulate DNA supercoiling and also can remove DNAknots and tangles.

TOPOISOMERASE II
Lower eukaryotes and invertebrates encode only a single type II topoisomerase, topoisomerase II (35–38). In contrast, vertebrate species encode two closely related Nuclear processes induce changes in DNA topology. DNA replication is used as an example. Although chromosomal DNA is globally underwound in all cells, the movement of DNA tracking systems generates positive supercoils. As shown in (A) chromosomal DNA ends are tethered to membranes or the chromosome scaffold (represented by the red spheres) and are unable to rotate. Therefore, the linear movement of tracking systems (such as the replication machinery represented by the yellow bars) through the immobilized double helix
compresses the turns into a shorter segment of the genetic material and
induces acute overwinding (i.e. positive supercoiling) ahead of the fork
(B). In addition, the compensatory underwinding (i.e. negative supercoiling)
behind the replication machinery allows some of the torsional stress that accumulates in the prereplicated DNA to be translated to the newly replicated daughter molecules in the form of precatenanes (C). If these precatenanes are not resolved, they ultimately lead to the formation of intertwined (i.e. tangled) duplex daughter chromosomes. Adapted from ref. 10. Nucleic Acids Research, 2009, Vol. 37, No. 3 739 isoforms of the enzyme, topoisomerase IIa and topoisomerase IIb. These isoforms differ in their protomer molecular masses (170 versus 180 kDa, respectively) and are encoded by separate genes (8,10,22,28,39–46).

Topoisomerase IIa and topoisomerase IIb display a high degree (70%) of amino acid sequence identity and similar enzymological characteristics. One notable difference between the two isoforms is that topoisomerase IIa relaxes (i.e. removes) positive superhelical twists 10 times faster than it does negative in vitro, while the b isoform is unable to distinguish the geometry of DNA supercoils during DNA relaxation (47). Topoisomerase IIa and topoisomerase IIb have distinct patterns of expression and separate cellular functions. Topoisomerase IIa is essential for the survival of proliferating cells, and protein levels rise dramatically during periods of cell growth (48–51). The enzyme is further
regulated over the cell cycle, with protein concentrations peaking in G2/M (50,52,53). Topoisomerase IIa is associated with replication forks and remains tightly bound to chromosomes during mitosis (9,51,54–56). Thus, it is believed to be the isoform that functions in growth-related processes, such as DNA replication and chromosome segregation (10,51). Topoisomerase IIb is dispensable at the cellular level but appears to be required for proper neural development (57–59).

Expression of topoisomerase IIb is independent of proliferative status and cell cycle, and the enzyme dissociates from chromosomes during mitosis (54,60,61). Topoisomerase IIb cannot compensate for the loss of topoisomerase IIa in mammalian cells, suggesting that these two isoforms do not play redundant roles in replicative processes (51,60,62,63). Although the physiological functions of topoisomerase IIb have yet to be defined, recent evidence indicates involvement in the transcription of hormonally or developmentally regulated genes (63,64). Much of what we understand regarding the mechanism of action of type II enzymes comes from experiments with topoisomerase II from species that express only a single form of the protein. Consequently, eukaryotic type II
topoisomerases will be referred to collectively as topoisomerase
II, unless the properties being discussed are specific to either the a or b isoform.
TOPOISOMERASE II-MEDIATED DNA CLEAVAGE
AND LIGATION
The ability of topoisomerase II to cleave and ligate DNA is central to all of its catalytic functions (5,8,11,27). All topoisomerases utilize active site tyrosyl residues to mediate DNA cleavage and ligation. Since type II enzymes cut both strands of the double helix, each protomer subunit contains one of these residues (Tyr805 and Tyr821 in human topoisomerase IIa and topoisomerase IIb,
respectively). Topoisomerase II initiates DNA cleavage by the nucleophilic
attack of the active site tyrosine on the phosphate of the nucleic acid backbone (Figure 2) (11,23,26,27). The resulting transesterification reaction results in the
formation of a covalent phosphotyrosyl bond that links the protein to the newly generated 50-terminus of the DNA chain.

It also generates a 30-hydroxyl moiety on the opposite terminus of the cleaved strand. The scissile bonds on the two strands of the double helix are staggered and are located across the major groove from one another. Thus, topoisomerase II generates cleaved DNA molecules with four-base 50-single-stranded cohesive ends, each of which is covalently linked to a separate protomer subunit of the enzyme (65–67). The covalent enzyme–DNA linkage plays two important roles in the topoisomerase II reaction mechanism. First, it conserves the bond energy of the sugar-phosphate DNA backbone. Second, because it does not allow the cleaved DNA chain to dissociate from the enzyme, the protein– DNA linkage maintains the integrity of the genetic material during the cleavage event (11,23,26,27). The covalent topoisomerase II-cleaved DNA reaction intermediate is referred to as the ‘cleavage complex’ and is critical for the pharmacological activities of the enzyme, which are discussed later in this article.

Although topoisomerase II acts globally, it cleaves DNA at preferred sites (68). The consensus sequence for cleavage is weak, and many sites of action do not
conform to it (68). Ultimately, the mechanism by which topoisomerase II selects DNA sites is not apparent, and it is nearly impossible to predict de novo whether a given DNA sequence will support scission. Most likely, the specificity of topoisomerase II-mediated cleavage is determined by the local structure, flexibility, or malleability of the DNA that accompanies the sequence, as opposed
to a direct recognition of the bases that comprise that sequence (69). Beyond the nucleophilic attack of the active site tyrosine on the DNA backbone, the details of topoisomerase II mediated DNA cleavage are not well defined.

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