TOPOISOMERASES: CHARACTERISTICS, FUNCTIONS, TYPES AND INHIBITORS - BIOLOGY - 2024

The topoisomerases are enzymes isomerases a type of modifying the topology of deoxyribonucleic acid (DNA), generating both its unwinding and supercoiling as its curl.

These enzymes have a specific role in relieving torsional stress on DNA so that important processes such as its replication, transcription of DNA into messenger ribonucleic acid (mRNA), and recombination of DNA can occur.

Figure 1. Topoisomerase II. Source: Emw, from Wikimedia Commons

Topoisomerase enzymes are present in both eukaryotic and prokaryotic cells. Its existence was predicted by scientists Watson and Crick, when evaluating the limitations that the structure of DNA presented to allow access to its information (stored in its nucleotide sequence).

To understand the functions of topoisomerases, it must be considered that DNA has a stable double-helix structure, with its strands wound one over the other.

These linear chains are made up of 2-deoxyribose linked by 5'-3 'phosphodiester bonds, and nitrogenous bases inside, like the rungs of a spiral staircase.

Figure 2. DNA molecule. Source:

The topological study of DNA molecules has shown that they can assume various conformations dependent on their torsional stress: from a relaxed state to different coiling states that allow their compaction.

DNA molecules with different conformations are called topoisomers. Thus, we can conclude that topoisomerases I and II can increase or decrease the torsional stress of DNA molecules, forming their different topoisomers.

Among the possible DNA topoisomers, the most common conformation is the supercoil, which is very compact. However, the DNA double helix must also be unwound by topoisomerases during various molecular processes.

characteristics

General mechanism of action

Some topoisomerases can relax only negative supercoils of DNA, or both supercoils of DNA: positive and negative.

If the circular double-stranded DNA is uncoiled on its longitudinal axis and a left-handed turn (clockwise) occurs, it is said to be negatively supercoiled. If the turn is clockwise (counterclockwise), it is positively overcoiled.

Figure 3. Negatively supercoiled, relaxed and positively supercoiled circular double-stranded DNA. Source: Fdardel, from Wikimedia Commons

Basically, topoisomerases can:

-Facilitate the passage of a DNA strand through a cut in the opposite strand (type I topoisomerase).

-Facilitate the passage of a complete double helix through a cleavage itself, or through a cleavage in another different double helix (type II topoisomerase).

In summary, topoisomerases act through the cleavage of phosphodiester bonds, in one or in both strands that make up DNA. They then modify the coiling state of the strands of a double helix (topoisomerase I) or of two double helices (topoisomerase II), to finally tie or tie the cleaved ends again.

Topoisomerases and the cell cycle

Although topoisomerase I is an enzyme that exhibits higher activity during S phase (DNA synthesis), it is not considered to be dependent on a phase of the cell cycle.

Whereas topoisomerase II activity is more active during the logarithmic phase of cell growth and in cells of fast-growing tumors.

Features

The alteration of the genes that code for topoisomerases is lethal for cells, evidencing the importance of these enzymes. Among the processes in which topoisomerases participate, are:

Compact storage of genetic material

Topoisomerases facilitate the storage of genetic information in a compact way, because they generate the coiling and supercoiling of DNA, allowing a large amount of information to be found in a relatively small volume.

Access to genetic information

Without topoisomerases and their unique characteristics, access to information stored in DNA would be impossible. This is due to the fact that topoisomerases periodically release the torsional stress that is generated in the DNA double helix, during its unwinding, in the processes of replication, transcription and recombination.

Figure 4. DNA replication. See topoisomerase at the beginning of the DNA hairpin. Source: LadyofHats translated by Miguelsierra, via Wikimedia Commons

If the torsional stress generated during these processes is not released, defective gene expression could occur, interruption of the circular DNA or chromosome, even producing cell death.

Regulation of gene expression

Conformational changes (in the three-dimensional structure) of the DNA molecule expose specific regions to the outside, which can interact with DNA-binding proteins. These proteins have a regulatory function of gene expression (positive or negative).

Figure 5. Regulatory protein of gene expression, in this case it prevents the expression of certain genes. Zephyris at the English language Wikipedia

Thus, the coiling state of DNA, generated by the action of topoisomerases, affects the regulation of gene expression.

Particularities of topoisomerase II

Topoisomerase II is necessary for the assembly of chromatids, the condensation and decondensation of chromosomes, and the segregation of daughter DNA molecules during mitosis.

This enzyme is also a structural protein and one of the main constituents of the cell nucleus matrix during interphase.

Types of topoisomerases

There are two main types of topoisomerases depending on whether they are able to cleave one or two strands of DNA.

-Topoisomerases type I

Monomeric

Type I topoisomerases are monomers that alleviate negative and positive supercoils, which are produced by hairpin movement during transcription, and during replication and gene recombination processes.

Type I topoisomerases can be subdivided into type 1A and type 1B. The latter are those found in humans, and they are responsible for relaxing supercoiled DNA.

Tyrosine in its active site

Topoisomerase 1B (Top1B) is made up of 765 amino acids divided into 4 specific domains. One of these domains has a highly conserved area containing the tyrosine active site (Tyr7233). All topoisomerases present a tyrosine in their active site with a fundamental role in the entire catalytic process.

Mechanism of action

The active site tyrosine forms a covalent bond with the 3'-phosphate end of the DNA strand, cutting it and holding it attached to the enzyme, while another strand of DNA passes through the cleavage.

The passage of the other DNA strand through the split strand is achieved thanks to a conformational transformation of the enzyme, which produces the opening of the DNA double helix.

Then topoisomerase I returns to its initial conformation and binds the cleaved ends again. This occurs by a process inverse to the breakdown of the DNA chain, at the catalytic site of the enzyme. Finally, topoisomerase releases the DNA strand.

The rate of DNA ligation is higher than the rate of excision, thereby ensuring the stability of the molecule and the integrity of the genome.

In summary, type I topoisomerase catalyzes:

1. The cleavage of a strand.
2. The passage of the other strand through the cleavage.
3. Ligation of the cleaved ends.

-Type II topoisomerases

Dimeric

Type II topoisomerases are dimeric enzymes, which cleave both strands of DNA, thus relaxing the supercoils that are generated during transcription and other cellular processes.

Mg dependent

These enzymes need magnesium (Mg ⁺⁺) and also need the energy that comes from breaking the ATP triphosphate bond, which they take advantage of thanks to an ATPase.

Two active sites with tyrosine

Human topoisomerases II are very similar to those of yeast (Saccharomyces cerevisiae), which is made up of two monomers (subfragments A and B). Each monomer has an ATPase domain, and in a sub-fragment the active site tyrosine 782, to which DNA can bind. Thus, two strands of DNA can bind to topoisomerase II.

Mechanism of action

The mechanism of action of topoisomerase II is the same as that described for topoisomerase I, considering that two DNA strands are split and not just one.

At the active site of topoisomerase II, a double-helix fragment of DNA, called "fragment G", is stabilized (through covalent bonding with tyrosine). This fragment is excised and held together to the active site by covalent bonds.

The enzyme then allows another DNA fragment, called the “T fragment”, to pass through the cleaved fragment “G”, thanks to a conformational change in the enzyme, which is dependent on the hydrolysis of ATP.

Topoisomerase II binds the two ends of the "G fragment" and finally recovers its initial state, releasing the "G" fragment. The DNA then relaxes the torsional stress, allowing replication and transcription to occur.

-Human topoisomerases

The human genome has five topoisomerases: top1, top3α, top3β (type I); and top2α, top2β (of type II). The most relevant human topoisomerases are top1 (type IB topoisomerase) and 2α (type II topoisomerase).

Topoisomerase inhibitors

-Topoisomerases as a target of chemical attack

Because the processes catalyzed by topoisomerases are necessary for the survival of cells, these enzymes are a good target of attack to affect malignant cells. For this reason, topoisomerases are considered important in the treatment of many human diseases.

Drugs that interact with topoisomerases are currently widely studied as chemotherapeutic substances against cancer cells (in different organs of the body) and pathogenic microorganisms.

-Types of inhibition

Drugs that inhibit topoisomerase activity can:

• Sandwiched in DNA.
• Affect the enzyme topoisomerase.
• Intercalate in a molecule close to the active site of the enzyme while the DNA-topoisomerase complex is stabilized.

Stabilization of the transient complex that is formed by the binding of DNA to the tyrosine of the catalytic site of the enzyme, prevents the binding of the cleaved fragments, which can lead to cell death.

-Topoisomerase inhibitor drugs

Among the compounds that inhibit topoisomerases are the following.

Antitumor antibiotics

Antibiotics are used against cancer, since they prevent the growth of tumor cells, usually interfering with their DNA. These are often called antineoplastic (cancer) antibiotics. Actinomycin D, for example, affects topoisomerase II and is used in Wilms tumors in children and rhabdomyosarcomas.

Anthracyclines

Anthracyclines are among the antibiotics, one of the most effective anticancer drugs and with the widest spectrum. They are used to treat cancer of the lung, ovaries, uterus, stomach, bladder, breast, leukemia, and lymphomas. It is known to affect topoisomerase II by intercalation in DNA.

The first anthracycline isolated from an actinobacteria (Streptomyces peucetius) was daunorubicin. Subsequently doxorubicin was synthesized in the laboratory, and epirubicin and idarubicin are also used today.

Anthraquinones

Anthraquinones or anthracenediones are compounds derived from anthracene, similar to anthracyclines, that affect topoisomerase II activity by intercalation in DNA. They are used for metastatic breast cancer, non-Hodgkin's lymphoma (NHL), and leukemia.

These drugs were found in the pigments of some insects, plants (frangula, senna, rhubarb), lichens and fungi; as well as in hoelite, which is a natural mineral. Depending on your dose, they can be carcinogenic.

Among these compounds, we have mitoxantrone and its analog, losoxantrone. These prevent the proliferation of malignant tumor cells, binding to DNA irreversibly.

Epidophyllotoxins

Podophyllotoxins, such as epidophyllotoxins (VP-16) and teniposide (VM-26), form a complex with topoisomerase II. They are used against lung cancer, testicular, leukemia, lymphomas, ovarian cancer, breast carcinoma and malignant intracranial tumors, among others. Podophyllum notatum and P. peltatum are isolated from plants.

Camptothecin analogs

Campothecins are compounds that inhibit topoisomerase I, including irinotecan, topotecan, and diflomotecan.

These compounds have been used against colon, lung and breast cancer, and are obtained naturally from the bark and leaves of the arboreal species Camptotheca acuminata of the Chinese and Tibet cornáceas.

Natural inhibition

Structural alterations of topoisomerases I and II can also occur completely naturally. This can happen during some events that affect your catalytic process.

These alterations include the formation of pyrimidine dimers, nitrogen base mismatches, and other events caused by oxidative stress.

References

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