Topoisomerases (or DNA 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.
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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. Topoisomerases can be further classified into subfamilies. In the type I family, there are two subfamilies; type IA and type IB when the enzyme links to the 5’ phosphate of the DNA strand, and the 3’ phosphate on the DNA, respectively. In the type II family, the structure and organism determine the subfamilies and their functions.
The overall function of DNA topoisomerase is to manage the topological state of the DNA in the cell. There are two types or families of this enzyme; type I family and type II family. Type I family passes one strand of the DNA through a break in the opposing strand. In other words, DNA topoisomerase type I enzyme cleaves only one strand of DNA. Type II family passes a region of duplex (two strands) from the same molecule or a different molecule through a double stranded gap. To summarize, type II cleaves both strands of DNA, that results in a double-stranded break. Topoisomerases can either relieve negative supercoils, both positive and negative supercoils, or induce positive and negative supercoiling in DNA. The enzymes also can promote catenation (when two single circular DNA strands are linked together after replication) and decatenation (the separation of two linked, closed, circular chromosomes), and can also relieve entanglement of linear chromosomes.
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 of animal cells 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.
Yeast cells are known to use three topoisomerases: Topoisomerase I, from the IB subfamily, is required for growth. It provides the replication fork with the ability to move forward, as well as removes positive and negative supercoils associated with transcription. Topoisomerase II from the IIA subfamily, is needed for decatenation of linked chromosomes and preparation for segregation during mitosis. Topoisomerase II cannot induce negative supercoils, but can relax both positive and negative supercoils like topoisomerase I, and can replace topoisomerase I if absent. Topoisomerase III from the IA family is used for cell growth. Without topoisomerase III, recombination rates in mitosis and meiosis can increase, which slows growth in cells. In S. pombe cells, III is used to sustain cell division.
Higher eukaryotic organisms are more complex organisms and typically require more complex cellular machinery. These organisms generally have a topoisomerase I, two type IIA topoisomerases, and two type III enzymes. Topoisomerase I helps with replication fork movement and relaxes supercoils associated with transcription. It is also used for relaxing solenoidal supercoils that form when chromosomes condense in preparation for mitosis. The two type IIA topoisomerases, IIα and IIβ, are used to unlink intertwined daughter duplexes, as well as assist in cell division and suppression of recombination, respectively. Type IIIα and IIIβ are thought to work in embryogenesis and interact with helicases, respectively.
E. coli contains four DNA topoisomerases: two type IA enzymes (I and III), and two type IIA (DNA gyrase and topoisomerase IV). DNA topoisomerase III and IV have similar functions. Topoisomerase III is incapable of relaxing positive supercoils, but it works to support replication fork movement on plasmid DNA in vitro. It can decatenate the winding that is happening behind the replication fork by focusing on nicks in the DNA. Topoisomerase IV is the most effective decatenating enzyme in E. coli. It also relaxes negative supercoils. DNA gyrase uses the hydrolysis of ATP to generate negative supercoiling in bacterial chromosomes. It relaxes positive supercoils ahead of the replication fork and acts in chromosome condensing. Finally, topoisomerase I helps with generating some negative supercoiling along with topoisomerase IV and DNA gyrase.
There is limited knowledge about the archaeal genome sequences. Therefore, there is also limited knowledge about the topoisomerase enzymes. They do contain a reverse gyrase, a type IA topoisomerase, and topoisomerase VI. The functions of the topoisomerases in archaea are comparable to the enzymes in eubacteria. The only noteworthy difference is that topoisomerase VI in archaea is responsible for decatenation of DNA replication intermediates, and it relaxes both positive and negative supercoils.
DNA topology is the tertiary conformations of DNA, such as supercoiling, knotting, and catenation. Topology of DNA can be disrupted by most metabolic processes: RNA polymerase can cause positive supercoils by over-winding the DNA in front of the enzyme, and can also cause negative supercoils by under-winding the DNA behind the enzyme. DNA polymerase has the same effect in DNA replication. Positive and negative supercoiling balance out the entire global topology of the DNA, so overall, the topology remains the same. However, as the DNA replication or transcription fork moves forward and positive supercoiling increases, the DNA strands wrap tighter and tighter around each other, making it more difficult for the polymerase to move forward. It is important for the local topology of DNA ahead of and behind the polymerase to be relieved so that replication and cell division can proceed. This is what DNA topoisomerases are used for.
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.
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.
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 that 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. In UniProt:, .||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||Homodimer|
|Role in suppressing recombination or supporting transcription in neurons||Homodimer|
|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.
DNA Topoisomerase Type I FamilyEdit
DNA Topoisomerase Type I family consists of two subfamilies; type IA and type IB. Type IA DNA topoisomerase amongst various organisms generally share the following properties: All of the enzymes are monomers. The enzyme shares a covalent interaction of a 5' phosphodiester bond at its tyrosine active site with the end of a DNA strand. The mechanism of relaxation of supercoiling requires magnesium(II). In plasmid DNA, the negative supercoils produced can be substrates for the relaxation mechanism, a process which does not go to completion. Type IA also requires an exposed single-stranded region within the DNA substrate. The linking number of DNA changes with relaxation. Type IA topoisomerase can catalyze catenation, decatenation, knotting and unknotting of the DNA.
There are three classes within the subfamily of type IB topoisomerase: topoisomerase I in eukaryotes, topoisomerase V in prokaryotes, and the poxvirus topoisomerase. Type IB subfamily topoisomerases are generally classified by their ability to relax both negative and positive supercoils, and the relaxation mechanism goes to completion (unlike type IA). They form a covalent interaction through the tyrosine active site on the enzyme and the 3’ phosphate on the DNA strand. The relaxation mechanism does not require magnesium(II). Both type IA and type IB topoisomerases, within the Type I family, have very distinct differences in their properties.
DNA Topoisomerase Type II FamilyEdit
Type II family of topoisomerases share general features and properties that make them distinguishable from the type I family. All type II DNA topoisomerases are dimers. They bind to a duplex DNA and cleave both strands, staggering four bases. Cleavage is done by a covalent interaction between each dimer subunit to the 5’ phosphate on the DNA, creating a phosphotyrosine bond. The reaction pulls the two ends of the cleaved DNA apart – this is called the gated (G-) segment. The transported (T-) segment, a region on the same or a different DNA duplex, is passed through G-segment. This changes the linking number when the DNA is circular (plasmid). The relaxation mechanism requires magnesium(II) and hydrolysis of ATP. The active site containing amino acid tyrosines have a helix-turn-helix motif, which collaborates with acidic residues to activate catalysis. Prokaryotic type II topoisomerases are heterotetrameric. Eukaryotic type II topoisomerases are homodimeric. Type II are also better at DNA relaxation, instead of decatenation, meaning they are better enzymes for relieving topological stresses in linear DNA versus circular DNA.
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