Topoisomerases are enzymes that participate in the overwinding or underwinding of DNA. The winding problem of DNA arises due to the intertwined nature of its double-helical structure. During DNA replication and transcription, DNA becomes overwound ahead of a replication fork. If left unabated, this torsion would eventually stop the ability of DNA or RNA polymerases involved in these processes to continue down the DNA strand.
In order to prevent and correct these types of topological problems caused by the double helix, topoisomerases bind to DNA and cut the phosphate backbone of either one or both the DNA strands. This intermediate break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed again. Since the overall chemical composition and connectivity of the DNA do not change, the DNA substrate and product are chemical isomers, differing only in their global topology, resulting in the name for these enzymes. Topoisomerases are isomerase enzymes that act on the topology of DNA.
Bacterial topoisomerases and human topoisomerases proceed via similar mechanisms for managing DNA supercoils.
The double-helical configuration of DNA strands makes them difficult to separate, which is required by helicase enzymes if other enzymes are to transcribe the sequences that encode proteins, or if chromosomes are to be replicated. In circular DNA, in which double-helical DNA is bent around and joined in a circle, the two strands are topologically linked, or knotted. Otherwise identical loops of DNA, having different numbers of twists, are topoisomers, and cannot be interconverted without the breaking of DNA strands. Topoisomerases catalyze and guide the unknotting or unlinking of DNA by creating transient breaks in the DNA using a conserved tyrosine as the catalytic residue.
The insertion of (viral) DNA into chromosomes and other forms of recombination can also require the action of topoisomerases.
Topologically linked circular molecules, aka catenanes, adopt a positive supercoiled form during the process of replication of circular plasmids. The unlinking of catenanes is performed by type IIA topoisomerases, which were recently found to be more efficient unlinking positive supercoiled DNA.The conformational properties of negative vs. positive supercoiled catenanes affects their features in respect to their corresponding enzymatic reaction catalyzed by topoisomerases. Experiments have demonstrated that positive supercoiled DNA provides a sharp DNA bend in the first bound DNA segment, which allows the topoisomerase to bind successfully and therefore carry on its enzymatic reaction to the following segment in a specific inside-to-outside matter. On the other hand, negative supercoiled DNA does not provide such bend and the access of the enzyme to the first segment is nearly impossible, therefore inhibiting unlinking.
Topoisomerase is also found in the mitochondria of cells. The mitochondria generates ATP as well as plays a role in programmed cell death and aging. The mitochondrial DNA is a circular, double-stranded DNA that requires the activity of topoisomerase to be replicated. The classes of topoisomerase found in the mitochondria are I, IIβ, IIIα.
Many drugs operate through interference with the topoisomerases The broad-spectrum fluoroquinolone antibiotics act by disrupting the function of bacterial type II topoisomerases. These small molecule inhibitors act as efficient anti-bacterial agents by hijacking the natural ability of topoisomerase to create breaks in chromosomal DNA.
Some chemotherapy drugs called topoisomerase inhibitors work by interfering with mammalian-type eukaryotic topoisomerases in cancer cells. This induces breaks in the DNA that ultimately lead to programmed cell death (apoptosis). This DNA-damaging effect, outside of its potential curative properties, may lead to secondary neoplasms in the patient.
There are three main types of topology:
Outside of the essential processes of replication or transcription, DNA must be kept as compact as possible, and these three states help this cause. However, when transcription or replication occurs, DNA must be free, and these states seriously hinder the processes. In addition, during replication, the newly replicated duplex of DNA and the original duplex of DNA become intertwined and must be completely separated in order to ensure genomic integrity as a cell divides. As a transcription bubble proceeds, DNA ahead of the transcription fork becomes overwound, or positively supercoiled, while DNA behind the transcription bubble becomes underwound, or negatively supercoiled. As replication occurs, DNA ahead of the replication bubble becomes positively supercoiled, while DNA behind the replication fork becomes entangled forming precatenanes. One of the most essential topological problems occurs at the very end of replication, when daughter chromosomes must be fully disentangled before mitosis occurs. Topoisomerase IIA plays an essential role in resolving these topological problems.
Topoisomerases can fix these topological problems and are separated into two types depending on the number of strands cut in one round of action: Both these classes of enzyme utilize a conserved tyrosine. However these enzymes are structurally and mechanistically different. For a video of this process click here.
- A type I topoisomerase cuts one strand of a DNA double helix, relaxation occurs, and then the cut strand is re-ligated. Cutting one strand allows the part of the molecule on one side of the cut to rotate around the uncut strand, thereby reducing stress from too much or too little twist in the helix. Such stress is introduced when the DNA strand is "supercoiled" or uncoiled to or from higher orders of coiling. Type I topoisomerases do not require ATP for hydrolysis are subdivided into three subclasses:
- Type IA topoisomerases, which share many structural and mechanistic features with the type II topoisomerases. Examples of type IA topoisomerases include prokaryotic Topoisomerase I and III, eukaryotic Topoisomerase IIIα and Topoisomerase IIIβ and Reverse Gyrase. Like type II topoisomerases, type IA topoisomerases form a covalent intermediate with the 5' end of DNA.
- Type IB topoisomerases, which utilize a controlled rotary mechanism. Examples of Type IB topoisomerases include Eukaryotic and eukaryal viral Topoisomerase I. In the past, type IB topoisomerases were referred to as eukaryotic topoisomerase I, but IB topoisomerases are present in all three domains of life. Type IB topoisomerases form a covalent intermediate with the 3' end of DNA.
- Type IC topoisomerase (also called Topoisomerase V) has been identified. While it is structurally unique from type IA and IB topoisomerases, It shares a similar mechanism with type IB topoisomerase.
- A type II topoisomerase cuts both strands of one DNA double helix, passes another unbroken DNA helix through it, and then re-ligates the cut strands. Type II topoisomerases utilize ATP hydrolysis and are subdivided into two subclasses which possess similar structure and mechanisms:
|Topoisomerase||Subfamily Type||Function||Multimericity||Metal Dependence||ATP Dependence||Single-or Double-Stranded Cleavage?||Cleavage Polarity||Change In Link Number (L)|
|Type IA||Removes (-), but not (+) supercoils||Monomer||Yes (Mg2+)||No||SS||5'||±1|
|Removes (-), but not (+) supercoils; Overlapping function with Topoisomerase IV|
|Removes (-), but not (+) supercoils; Assists in the unlinking of precatenanes in cellular DNA replication; Can catalyze the knotting, unknotting, and interlinking of single-stranded circles as well as the knotting, unknotting, catenation, and decatenation of gapped or nicked duplex DNA circles.|
|Reverse DNA Gyrase
|Removes (-), but not (+) supercoils||Heterodimer|
|Reverse DNA Gyrase
|Removes (-), but not (+) supercoils|
|Type IB||Remove (+) and (-) supercoils; Relaxes compensatory (-) supercoils; Generates right-handed solenoidal supercoils; Supports fork movement during replication; Thought to be similar in structure to tyrosine recombinases.||Monomer||No||No||SS||3'||±1|
|Type IC||Relaxes (+) and (-) supercoils. Involved in DNA repair||Monomer||No||No||SS||3'||±1|
|Topoisomerase II / DNA Gyrase
|Type IIA||Generates (-) supercoils (the only topoisomerase known to do this)||Heterotetramer||Yes (Mg2+)||Yes||DS||5'||±2|
|Relaxes (-) supercoils; Role in decatenation||Heterotetramer|
|Essential; Unlinks intertwined daughter duplexes in replication; Contributes to DNA relaxation during transcription||Heterodimer|
|Role in suppressing recombination or supporting transcription in neurons||Heterodimer|
|Type IIB||Relaxes (+) and (-) supercoils; Responsible for decatenating replication intermediates; May be exclusive to the archaea.||Heterotetramer||Yes (Mg2+)||Yes||DS||5'||±2|
Both type I and type II topoisomerases change the linking number (L) of DNA. Type IA topoisomerases change the linking number by one, type IB and type IC topoisomerases change the linking number by any integer, whereas type IIA and type IIB topoisomerases change the linking number by two.
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