DNA Topology and Topoisomerases: Teaching a "Knotty" Subject

Abstract

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.

Fig. 1. Demonstrating topological relationships with a balloon

The relationship between the inside and outside of a balloon is topological in nature. Despite stretching and distorting the balloon by inflation (or even by creating balloon animals for the adventurous lecturer), the inside and outside never meet. In order to alter this topological relationship, the “closed system” of the balloon must be disrupted. This can be accomplished in dramatic fashion by using a pin. Once the topological system is opened, the inside and the outside of the balloon can touch.

Fig. 2. Topological relationships within DNA

DNA molecules are shown as circular ribbons for simplicity. Top: DNA with no torsional stress is referred to as “relaxed.” Underwinding or overwinding DNA results in negative (−) or positive (+) supercoils, respectively. The directionality of the DNA is shown by internal arrowheads in the negatively supercoiled [(−)SC] molecule. Supercoils are shown as writhes (DNA crossovers or nodes) for visual ease, but it should be noted that supercoils can be interconverted from writhes to twists. By convention, each writhe (denoted by the crossing of one DNA segment over another segment) is given an integral value of −1 or +1. Middle: Tracing the direction of the DNA, the sign of the node is assigned based on the direction of movement required to align the front segment of DNA with the back segment using a rotation of <180° [5]. Negative DNA writhes are represented by crossovers in which the front segment must be rotated in a clockwise manner to align it with the back segment, while positive writhes require a counterclockwise rotation. Bottom: Intramolecular knots and intermolecular tangles also form in DNA.

Fig. 3. Movement of DNA tracking systems generates positive supercoils

The ends of chromosomal DNA are anchored to membranes or the chromosome scaffold (represented by the red spheres) and are not free to rotate. Therefore, the linear movement of tracking systems (such as replication machinery represented by the yellow bars) through the double helix does not change the number of turns of the DNA. This action merely compresses the turns into a shorter segment of the genetic material. Consequently, the double helix becomes increasingly overwound, generating positive (+) supercoils ahead of tracking systems. Adapted from Ref. .

Fig. 4. DNA relaxation catalyzed by type II topoisomerases removes two supercoils per double-stranded DNA passage event

Type II topoisomerases relax DNA using a sign inversion mechanism. The double-stranded DNA passage reaction inverts the sign of a writhe (crossover). In the example shown, a negative writhe is converted to a positive writhe, changing the ΔLk from −2 to 0.

Fig. 5. Topoisomerases are essential but genotoxic enzymes

The formation of topoisomerase-DNA cleavage complexes is required for the enzymes to perform their critical cellular functions. If the level of topoisomerase II-DNA cleavage complexes falls too low (left arrow), cells are unable to segregate chromosome and ultimately die of mitotic failure. If the level of cleavage complexes (generated by topoisomerase I or II) becomes too high (right arrow) the actions of DNA tracking systems can convert these transient complexes to permanent double-stranded breaks. The resulting DNA breaks, as well as the inhibition of essential DNA processes, initiate recombination/repair pathways and generate chromosome translocations and other DNA aberrations. If the strand breaks overwhelm the cell, they can trigger apoptosis (or the SOS response in bacteria). This is the basis for the actions of several widely prescribed anticancer (and antibacterial) drugs. If the concentration of topoisomerase-mediated DNA strand breaks is too low to overwhelm the cell, mutations or chromosomal aberrations may be present in surviving populations. In some cases, exposure to topoisomerase II poisons has been associated with the formation of specific types of leukemia that involve the MLL (mixed lineage leukemia) gene at chromosome band 11q23 or the chromosome 15;17 translocation that joins the PML (promyelocytic leukemia) and RARA (retinoic acid receptor α) genes (lower right arrow).

Fig. 6. Topoisomerase poisons

Structures of selected topoisomerase I-targeted anticancer drugs, topoisomerase II-targeted anticancer drugs, naturally occurring topoisomerase II poisons, and quinolone antibacterials are shown.