DNA topoisomerase II, genotoxicity, and cancer

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Abstract

Type II topoisomerases are ubiquitous enzymes that play essential roles in a number of fundamental DNA processes. They regulate DNA under- and overwinding, and resolve knots and tangles in the genetic material by passing an intact double helix through a transient double-stranded break that they generate in a separate segment of DNA. Because type II topoisomerases generate DNA strand breaks as a requisite intermediate in their catalytic cycle, they have the potential to fragment the genome every time they function. Thus, while these enzymes are essential to the survival of proliferating cells, they also have significant genotoxic effects. This latter aspect of type II topoisomerase has been exploited for the development of several classes of anticancer drugs that are widely employed for the clinical treatment of human malignancies. However, considerable evidence indicates that these enzymes also trigger specific leukemic chromosomal translocations. In light of the impact, both positive and negative, of type II topoisomerases on human cells, it is important to understand how these enzymes function and how their actions can destabilize the genome. This article discusses both aspects of human type II topoisomerases.

Introduction

Perhaps the most striking feature of DNA is the intertwining of the two complementary strands of the double helix [1]. Discovery of this characteristic led to the immediate recognition that biological processes such as replication would be severely affected by the topological state of the genetic material [2].

DNA is globally underwound (i.e., negatively supercoiled) in all species ranging from eubacteria to humans [3], [4], [5], [6]. This underwinding makes it easier to separate complementary DNA strands from one another and greatly facilitates initiation and elongation of replication and transcription. Once the replication or transcription machinery begins to travel along the DNA template, however, deleterious effects of topology manifest themselves. Since helicases separate, but do not unwind the two strands of the double helix, fork movement results in acute over-winding (i.e., positive supercoiling) of the DNA ahead of the tracking systems [3], [5], [6], [7]. In contrast to underwinding, overwinding dramatically increases the difficulty of separating duplex DNA into individual strands. Therefore, accumulation of positive supercoils presents a formidable block to replication, transcription, and other essential DNA processes [5], [7], [8], [9], [10].

In addition to issues related to DNA under/overwinding, nuclear processes such as recombination and replication generate knots and tangles in the genetic material. If knots accumulate in the genome, DNA tracking systems are unable to separate the two strands of the double helix [3], [5], [6], [7], [11]. Moreover, if tangled (i.e., catenated) daughter chromosomes are not resolved prior to cell division, cells will die of mitotic failure [7], [12], [13], [14], [15], [16].

The topological state of DNA in the cell is modulated by enzymes known as topoisomerases [5], [7], [12], [13], [14], [15], [16], [17], [18], [19]. These ubiquitous enzymes regulate DNA over- and underwinding, and remove knots and tangles from the genetic material by creating transient breaks in the sugar-phosphate backbone of the double helix [5], [7], [12], [13], [14], [15], [16], [17], [18], [19]. Topoisomerases maintain genomic integrity during this process by forming covalent attachments between active site tyrosyl residues and the terminal DNA phosphates that are generated during the cleavage reaction [5], [12], [14], [15], [16], [17], [18], [19], [20]. This covalent linkage is the hallmark characteristic of all DNA topoisomerases.

Cells encode two classes of topoisomerases that are distinguished by their catalytic mechanisms. Type I topoisomerases act by generating a transient single-stranded break in the double helix, followed by either a single-stranded DNA passage event or controlled rotation about the break [5], [12], [14], [18], [21], [22]. As a result, these enzymes are able to alleviate torsional stress (i.e., remove superhelical twists) in duplex DNA. Type I topoisomerases are involved in all DNA processes that involve tracking systems and play important roles in maintaining genomic integrity [5], [7], [13], [14], [18], [21], [22].

Type II topoisomerases act by generating a transient double-stranded DNA break, followed by a double-stranded DNA passage event [14], [15], [16], [19]. Consequently, these enzymes are able to remove superhelical twists from DNA and resolve knotted or tangled duplex molecules. Type II topoisomerases function in numerous DNA processes and are required for recombination, the separation of daughter chromosomes, and proper chromosome structure, condensation, and decondensation [5], [6], [7], [12], [13], [14], [15], [16].

Section snippets

Topoisomerase II isoforms

Whereas lower eukaryotes such as yeast and Drosophila encode only a single type II topoisomerase [23], [24], vertebrate species express two discrete forms of the enzyme, topoisomerase IIα and IIβ [14], [19], [25], [26]. These enzymes display a high degree of amino acid sequence identity (∼70%), however, they differ in their protomer molecular masses (170 kDa versus 180 kDa, respectively) and are encoded by separate genes [7], [14], [15], [16], [19], [25], [26], [27], [28], [29], [30].

Topoisomerase II as a genotoxic enzyme

Topoisomerase II–DNA cleavage complexes are transient in nature and their cellular concentration is tightly regulated (Fig. 2). Cleavage complex formation is essential for topoisomerase II to perform its cellular functions [5], [14], [15], [16], [19]. If the level of topoisomerase II–DNA cleavage complexes falls too low (i.e., enzyme activity is lowered), cells are unable to undergo chromosome segregation and ultimately die of mitotic failure [7], [12], [13], [14], [15], [16].

Although the

Topoisomerase II poisons as anticancer agents and cellular toxins

Agents that increase levels of topoisomerase II–DNA cleavage complexes are known as “topoisomerase II poisons” because they convert this essential enzyme to a potent cellular toxin. Topoisomerase II poisons increase levels of enzyme–DNA cleavage complexes by two non-mutually exclusive mechanisms [15], [16], [79], [80], [81]. Some poisons act by inhibiting the ability of topoisomerase II to ligate the cleaved substrate [15], [16], [81], [100]. These agents not only increase the level of cleavage

Effects of DNA supercoiling on topoisomerase II-mediated DNA cleavage and the actions of anticancer drugs

As discussed above, topoisomerase II–DNA cleavage complexes are transient in nature and are converted to permanent DNA strand breaks in the cell when replication or transcription complexes (or other nucleic acid tracking systems) collide with the covalently attached enzyme [15], [16], [78], [79]. It is these permanent DNA strand breaks that ultimately initiate the cytotoxic effects of topoisomerase II poisons [83].

Globally, DNA in all eukaryotic cells is underwound (i.e., negatively

Conclusions

Type II topoisomerases are ubiquitous enzymes that play essential roles in a number of critical DNA processes. In addition, they are the cytotoxic targets for a number of highly successful anticancer drugs. Despite the significance of topoisomerase IIα and IIβ to the survival of human cells and the efficacy of cancer chemotherapy, considerable evidence indicates that these enzymes have significant genotoxic effects and can trigger specific leukemic chromosomal translocations. In light of the

Acknowledgements

Work in the senior author's laboratory was supported by National Institutes of Health research grants GM33944 and GM53960. AKM was a trainee under National Institutes of Health grant 5 T32 CA09582.

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