Topoisomerase

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Functioning of the E.Coli Girasa DNA. Type II Topoisomerase

The topoisomerases (type I: EC 5.99.1.2, and type II: EC 5.99.1.3) are enzymes capable of acting on the topology of DNA, either by entangling it to allow it to be stored more compactly or untangle it to control protein synthesis and to facilitate its replication. These enzymes are necessary because of the inherent problems caused by the structural configuration of DNA.

DNA has a structure in the form of a double helix in which two strands or chains of 2-deoxyribose sugar linked by hydrogen bonds are wound one over the other, with four bases, adenine, thymine, cytosine and guanine paired and Stacked in the central hole of this helix almost like the steps on a spiral staircase, this structure allows genetic material to be preserved beautifully and stably. But Watson and Crick had already noticed that the two DNA strands were coiled and twisted around each other, and that this would necessarily require a mechanism capable of developing and separating the strands to allow access to the stored information.

In order to get around the problems caused by this double helix configuration, topoisomerases are able to cut and paste either one or two of the sugar phosphate strands that form the framework or backbone of DNA. This selective cleavage allows the DNA to unravel and unwind to allow other enzymes access to the information contained within. At the end of the process, the DNA is restored to its initial configuration. And since tangled and untangled DNA have the same chemical composition and the same bonds arranged in the same way, the two forms of DNA, tangled and untangled, are isomers that differ only in their overall configuration. Hence the name of the enzymes. Topoisomerases are isomerase enzymes that act on the topology of DNA.

Discovery

The need for this enzyme was recognized even before it was discovered, when the double-helical nature of DNA was resolved by Watson and Crick; the authors noted that there must be some mechanism that could resolve or overcome the entanglements that would arise from this structural feature. The first disclosed topoisomerase, E. coli topo I, was discovered by James C. Wang.

Function

The stability derived from the double helix configuration of DNA naturally resides in the fact that, thanks to this configuration, it is very difficult to separate the two strands, making the action of helicases or other enzymes essential to allow the transcription of those sequences. DNA that code for proteins, or even if chromosomes are to be replicated. In the case of circular chromosomes such as those found in bacteria, in which the double helix is joined at the ends, literally forming a closed circle, the two strands are topologically coiled, or knotted. That is to say, identical loops of DNA but with different numbers of turns on itself are topoisomers, and cannot be interconverted by any process that does not involve breaking the two DNA strands. Topoisomerases catalyze and guide the untying and stretching of DNA by creating momentary breaks in the DNA strands and using an evolutionarily highly conserved tyrosine as the catalytic residue.

Insertion of a viral genome into chromosomes and other forms of genetic recombination also require the action of topoisomerases.

Clinical significance

See also Topoisomerase inhibitor

Many drugs work by interfering with topoisomerases [3]. All broad-spectrum antibiotics of the fluoroquinolone family, eg ciprofloxacin, work by interfering with the function of bacterial type II topoisomerases, causing uncontrollable breaks in DNA strands due to the build-up of internal stresses caused by the curls. These small inhibitory molecules act as efficient bactericides by simply disrupting topoisomerase's natural ability to relieve structural stresses by creating controlled breaks in DNA strands.

Some types of chemotherapy drugs called topoisomerase inhibitors work by interfering with mammalian eukaryotic topoisomerases in cancer cells. Since topoisomerases also fulfill the structural function of relieving stresses caused by DNA coiling, inhibition of eukaryotic topoisomerases induces DNA breaks that ultimately force cells into the cycle of programmed cell death (apoptosis). However, this effect of DNA damage can cause secondary neoplasms in the people treated, so the cost-benefit ratio is taken into account in this type of treatment.

Topoisomerase I is the antigen that is recognized by Anti Scl-70 antibodies in the autoimmune disease scleroderma.

Topological problems

DNA has three main types of topology: supercoiling, knotting, and concatenation. Apart from the essential processes of replication or transcription, the rest of the time the DNA must be kept in as compact a configuration as possible, and these three topological states contribute to the cause. However, when transcription or replication processes occur, the DNA must be separated into single strands, and coiled states seriously hinder this process. And additionally, during replication, the newly replicated DNA duplex and the original strand end up entangled with each other, and must be completely separated to ensure genome integrity while the cell divides. As the transcriptional bubble travels down the strand, the DNA ahead of the transcription fork becomes supercoiled, while the DNA downstream of the fork becomes nearly uncoiled. When DNA replication occurs, the double strand ahead of the replication fork becomes positively supercoiled, while the DNA behind the fork becomes entangled, forming precatenans. One of the most fundamental topological problems occurs at the end of replication, when the daughter chromosomes must be completely untangled before mitosis can take place. Type II topoisomerase plays an essential role in solving these topological problems, plus it has extra holes.

Types of Topoisomerases

Topoisomerases can solve DNA topological problems, and based on the type of mechanism they use they can be separated into two large groups based on the number of DNA strands they cut per round of action: both types of enzymes use an evolutionarily highly conserved tyrosine residue as the active site, however both types of enzymes are structurally and mechanistically different.

  • The Topoisomerase type I cuts a strand of DNA, thus allowing to release internal tensions due to excessive rolling or poor rolling. Once the tension has relaxed, stick the ends of the cut strand. By cutting one of the strands, the molecule is allowed to rotate around the link that remains full by reducing stress due to the enrollment. Type I topoisomerase does not require ATP for operation. Type I topoisomerase are frequently subdivided into two subclasses: IA Topoisomerase; which have many structural and mechanistic similarities with type II topoisomerase. And IB topoisomerase; which use a controlled rotation mechanism.

Some examples of type IA topoisomerases include topo I and topo III. In the past type IB topoisomerases were often referred to as eukaryotic type I topoisomerases, but type IB topoisomerases are present in all three domains of life. It is interesting to note that type IA topoisomerases form a covalent intermediate with the 5' phosphate end (leaving a free 3' OH) of the single stranded DNA, while type IB topoisomerases do so with the 3'phosphate (leaving a free 5'OH).

Recently, a new type of isomerase I(IC) has been identified, called topo V. Despite being totally different from topoisomerases IA and IB from a structural point of view, it shares similarities with the mechanism of topoisomerases IB:

  • The type II topoisomerase cuts both strands of the DNA chain, and passes another double chain intact by the gap formed in the rupture. These are also divided into two subclasses: IIA and IIB type, which are similar in structure and mechanism. Examples of IIA-type topoisomerase include topo II eucaritic, the E. coli toura, and the E. coli topo IV. An example of IIB-type toposiomerase would be topo VI.
TopoisomerasaIAIBIIAIIB
Metal DependentYeah.No.Yeah.Yeah.
ATP DependantsNo.No.Yeah.Yeah.
Single (SS) or Double (DS) yarnSSSSDSDS
Rupture Polarity5'3'5'5'
Change in NE±1±N±2±2

Both types of topoisomerases change the linking number (NE) of DNA. Type IA topoisomerases change the binding number by one, types IB and IC cause a change by one integer, while type IIA and IIB topoisomerases cause a change of two in the binding number.

Further reading

  • James C. Wang (2009) Untangling the Double Helix. DNA Entanglement and the Action of the DNA TopoisomerasesCold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2009. 245 pp. ISBN 978-0-87969-879-9

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