What is Topoisomerases?
Topoisomerases are nuclear enzymes that play essential roles in DNA replication, transcription, chromosome segregation, and recombination.
All cells have two major forms of topoisomerases: type I, which makes single-stranded cuts in DNA, and type II enzymes, which cut and pass double-stranded DNA. DNA topoisomerases are important targets of approved and experimental anti-cancer agents.
DNA topoisomerases (or topoisomerases) are enzymes that catalyze changes in the topological state of DNA, interconverting relaxed and supercoiled forms, linked (catenated) and unlinked species, and knotted and unknotted DNA.
Topological issues in DNA arise due to the intertwined nature of its double-helical structure, which, for example, can lead to overwinding of the DNA duplex during DNA replication and transcription.
If left unchanged, this torsion would eventually stop the DNA or RNA polymerases involved in these processes from continuing along the DNA helix. A second topological challenge results from the linking or tangling of DNA during replication.
Left unresolved, links between replicated DNA will impede cell division. The DNA topoisomerases prevent and correct these types of topological problems.
They do this by binding to DNA and cutting the sugar-phosphate backbone of either one (type I topoisomerases) or both (type II topoisomerases) of the DNA strands.
This transient break allows the DNA to be untangled or unwound, and, at the end of these processes, the DNA backbone is resealed.
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 topology.
Types of Topoisomerases
Topoisomerases transiently break one or both DNA strands, classifying the enzymes into 2 types: Type I topoisomerase and Type II topoisomerase.
Type I Topoisomerases
Type I topoisomerase is a type of topoisomerase that cuts on a single strand of DNA. It is not an ATP-dependent enzyme(exception: Reverse Gyrase).
It mainly changes the linking number by plus one.
Note: Odd types of topoisomerases come under type I and even types under type II.
Type I Topoisomerase Structure
There is the presence of multiple varying domains in the type IA. It can be from I to IV. Toprim domain is contained in domain I. HTH (Helix-Turn-Helix) is present in domains III and IV. The tyrosine residues are present in the HTH of domain III. It appears like a lock with all three domains present at bottom of the topoisomerase structure.
Type IB contains active site (tyrosine) bind with C-terminal domain, N-terminal domain, capping, and catalytic lobe.
Type I Topoisomerase Types
It is of three basic types:
Type IA topoisomerases
It binds to the 5′ Carbon end of the DNA.
This type of topoisomerase show homology to topoisomerase I of E. coli.
It is of further three types:
Topo IA: It is found in eubacteria.
Topo III: It is found in eubacteria and eukaryotes.
Reverse Gyrase: It is found in archaebacteria and eubacteria as well. It is the only type of type I topoisomerase that is ATP-dependent.
Type IB topoisomerases
It binds to the 3′ Carbon end of the DNA. It forms nick in one strand. This type of topoisomerase show homology to topoisomerase I of humans.
Type IC topoisomerases
It contains one type of topoisomerase i.e. topoisomerase V. It binds to the 3′ Carbon end of the DNA. It is found in archaebacterial. It shows the controlled mechanism of rotation.
Type I Topoisomerase Mechanism of action
It generally occurs in the following events occurring together at the same time.
Cutting a single strand of DNA: Active site of the topoisomerase contains an amino acid tyrosine. The disruption of phosphodiester bond and formation of intermediate with phospho-tyrinosyl linkage favors the breaking of a DNA strand. The bond formation and cleavage mechanism in detail are the same as in the case of type II topoisomerase. Tyrosine may attack 3’or 5’carbon end.
Passing of strand: After the cleavage, the uncut DNA strand passes through the break. In this step, the enzyme changes from closed conformation to open conformation favoring the passing of strand. No ATP is utilized in this conformational change in the case of type I.
Religation: The phosphate linked with tyrosine is again attacked by the OH of the ribose group of the strand which was separated before and it results in the removal of intermediate linkage of tyrosine and rejoining of the cleaved strand. The enzyme returns to its initial stage(closed conformation) and is recovered for the next cycle.
Type I Topoisomerase Functions
They are involved in the removal of supercoils of DNA in biological processes such as replication and transcription.
- Help in relaxing DNA.
- They help in breaking strands during recombination.
- They are also involved in the condensation of the chromosome.
- During mitosis, the DNA strands need to be free from interwinding which is done by topoisomerase I.
Type II Topoisomerase
Type II Topoisomerase Definition
Type II topoisomerase is a type of topoisomerase that cuts on both strands of DNA at once. It is an ATP-dependent enzyme. It changes the linking number by two.
Type II Topoisomerase Structure
Topoisomerase IIA in eukaryotes consists of two same monomers (A-A) whereas in prokaryotes they are formed heterotetramers (A2B2).
Topoisomerase IIB is formed of heterotetramers only.
Topoisomerase II consists of four domains which include:
- ATPase domain at N-terminal
- A variable C-terminal domain
- Domain for binding of DNA located centrally
- A conserved domain of about a hundred amino acids i.e. toprim domain.
Type II Topoisomerase Types
It is of two basic types:
Type IIA topoisomerases
It is found in viruses and all cellular organisms. It is of three types:
Topo II: It is found in eukaryotes.
Topo IV: It is found in bacteria. It differs from Gyrase. It is not involved in DNA wrapping while Gyrase is involved in DNA wrapping and promoting negative supercoils.
Gyrase: It is found in bacteria and some eukaryotes. It introduces negative supercoiling decreasing the linking number by two.
Type IIB topoisomerases
It includes Topo VI which can be found in archaea and some plants.
Type II Topoisomerase Mechanism of action
It occurs as follows with ATP hydrolysis.
Cleaving of DNA chain: The enzyme contains tyrosine residues. They form covalent bonds with the DNA strands and break the DNA chain. The lone pair of electrons of O-atom present in the tyrosine acts as a nucleophile and attacks on the Phosphorus in phosphate of DNA.
It causes the shifting of a bond from phosphate to one of the O-atom attached to the ribose sugar forming a hydroxyl group.
Hence the covalently bonded tyrosine attached with phosphorus breaks the phosphate-sugar backbone which cleaves the chain. This linking is termed 5′-phospho-tyrinosyl protein-DNA linkage.
A duplex is broken by the action of the enzyme on both strands at once.
Crossing of the intact strand through the gap: In this case, another whole duplex strand passes through the gap over the broken duplex. In this the conformational change in enzyme requires ATP.
Religation: It is done by the attack of 3′-OH of the sugar of separated strand on phosphate group which has formed an intermediate linkage with tyrosine. It repels the bond with tyrosine and reforms the broken bond to join again. It occurs on both strands of duplex together ligating them. The enzymes regain their conformation and continue the cycle.
Type II Topoisomerase Functions
- It increases the disentanglement of the chromosome.
- It does not aid in the supercoiling of DNA but is involved in their relaxation.
- DNA gyrase promotes the negative supercoils of DNA.
- One of the most important functions is that it brings the change of two units in the linking number of loops in DNA.
How Does Topoisomerase Work?
Different types of topoisomerases have different action mechanisms based on their structure.
DNA topo I work on the principle of the strand-passage mechanism. The catalytic events that occur include:
The topo enzyme contains a tyrosine residue in its active site which catalyzes DNA strand breaks (DNA cleavage) by forming a covalent bond with the phosphate group of DNA.
The uncut DNA strand passes through the DNA break, and at this position, topoisomerase changes its conformation from a closed to an open state.
The linkage between tyrosine and phosphate is broken, the bond forms between the nucleotides again, and the strands are rejoined. The enzyme again changes back to its closed state.
DNA topo II action mechanism involves ATP hydrolysis. The steps of the reactions are:
The enzyme-containing tyrosine forms a bond with the phosphate group of the nucleic acid and breaks the bonding between the two strands.
Another DNA double helix passes through the DNA breaks, and the topoisomerase enzyme changes its conformation, which requires ATP hydrolysis.
The 3’ OH group of the separated strands attacks the phosphate group of the strand that bonded with the tyrosine residue of the enzyme. This breaks the 5′-phospho-tyrinosyl protein-DNA bond and relegates two DNA strands. At this stage, the topo enzyme converts back to its original conformation.
Functions of Topoisomerase
Topoisomerase is important to DNA replication because it relaxes supercoiled DNA ahead of the replication fork so that replication can continue to occur.
There are a number of different types of topoisomerases, each specialising in a different aspect of DNA manipulation.
Accessing DNA
During transcription and DNA replication, the DNA needs to be unwound in order for the transcription/replication machinery to gain access to the DNA so it can be copied or replicate, respectively. Topoisomerase I can make single-stranded breaks to allow these processes to proceed.
Removing DNA Supercoils
During transcription and DNA replication, the DNA helix can become either over-wound or under-wound.
For instance, during DNA replication, the progress of the replication fork generates positive supercoils ahead of the replication machinery and negative supercoils behind it. Such tensional problems also exist when transcribing DNA to make an RNA copy for protein synthesis.
During these processes, the DNA can be supercoiled to such an extent that if left unchecked it could impede the progress of the protein machinery involved. This is prevented by topoisomerase I, which makes single-stranded nicks to relax the helix.
Strand Breakage during Recombination
Before the chromosomes separate from one another during cell division, they are able to exchange genetic information through a process known as recombination, where physical pieces of DNA on one chromosome can be swapped for DNA on the matching sister chromosome in order to shuffle the genetic information.
Topoisomerase III can introduce single-strand breaks that are required for DNA to be exchanged by adjacent chromosomes.
Chromosome Condensation
During the cell cycle, chromosomes must be alternatively condensed and decondensed at specific stages.
Topoisomerase II (gyrase) acts as a molecular motor, using the energy gained from ATP hydrolysis to introduce tight supercoils into the DNA helix in order to condense the chromosome.
Because this process must be highly regulated, topoisomerase II can form molecular complexes with important cell cycle regulators (such as p53, TopBP1, 14-3-3 epsilon, and Cdc2) to ensure that chromosome condensation occurs at the correct time in the cell cycle.
Disentangling Intertwined DNA
During cell division, once the chromosomes have been replicated, they must separate and travel to opposite ends of the cell to become part of two separate daughter cells.
Topoisomerases IV acts to disentangle the replicated daughter strands by making double-strand breaks that allow one duplex to pass through the other.
Topoisomerases as Drug Targets
Topoisomerases have been the focus for the treatment of certain diseases.
Bacterial gyrase (topoisomerase II) and topoisomerase IV are the targets of two classes of antibiotic drugs: quinolones and coumarins.
These antibiotics are used to treat an assortment of different diseases, such as pneumonia, tuberculosis and malaria, by inhibiting DNA replication in the bacteria responsible.
Eukaryotic topoisomerases I and II are the targets of an increasing number of anti-cancer drugs that act to inhibit these enzymes by blocking the reaction that reseals the breaks in the DNA.
Often the binding of the drug is reversible, but if a replication fork runs into the blocked topoisomerase, then a piece of the gapped DNA strand not bound by the topoisomerase could be released, creating a permanent breakage in the DNA that leads to cell death.
Most of these inhibitors are selective against either topoisomerase I or II, but some can target both enzymes.
Topoisomerase I inhibitors induce single-strand breaks into DNA, and can work by a variety of mechanisms.
Some drugs, such as camptothecins, inhibit the dissociation of topoisomerase and DNA, leading to replication-mediated DNA damage, which can be repaired more efficiently in normal cells than in cancer cells (deficient for DNA repair).
Topoisomerase I inhibitors can also cause gene inactivation through chromatid aberrations.
Topoisomerase II inhibitors, such as anthracyclines, are amongst the most widely used anti-cancer agents.
These drugs are potent inducers of double strand breaks in DNA, and can cause arrest in the cell cycle at the G2 stage, the latter occurring by disrupting the interaction between topoisomerase II and regulators of the cell cycle, such as Cdc2.
Topoisomerase II inhibitors can cause a wide range of chromosomal aberrations, and can act by either stabilising topoisomerase II-DNA complexes that are easily cleaved, or by interfering with the catalytic activity of the enzyme, both resulting in double-strand breaks in the DNA.
There are also dual inhibitors that target both topoisomerase I and II, which increases the potency of the anti-cancer effect.
These drugs work by a variety of means: by recognising structural motifs present on both enzymes, by linking separate topoisomerase inhibitors together into a hybrid drug, or by using inhibitors that bind to and intercalate DNA.