Topoisomerase: Overview & Applications

Two strands of rope unwinding and winding around one another

Topoisomerase: Overview

Topoisomerase (DNA topoisomerases) is an enzyme that catalyzes the changes in the intertwined state of two DNA strands. It helps in vivo and in vitro DNA replication, transcription, chromosome segregation, and recombination.

It mainly facilitates the interconversion forms (also known as topological changes) of relaxed and supercoiled DNA, knotting and unknotting DNA, and linked (catenate) and unlinked (decatenate) species.

Naturally, the topological changes occur either due to intertwining in the helical structure of DNA or linking/tangling of the DNA helix during genome replication. However, they are corrected by the enzyme topoisomerase.

If left uncorrected, these issues can hinder cell division, DNA replication, and transcription (by preventing DNA and RNA polymerase enzymes from continuing along the helix) processes.  

Topoisomerase was discovered in 1957 by the scientist J.C. Wang when he was working on Escherichia coli (or E.coli). Similar topoisomerase activity was observed by James Champoux and Renato Dulbecco in eukaryotic cells. 

The enzyme works by breaking the phosphodiester bond present in the backbone of the two DNA strands. However, the broken bonds in the DNA molecules reform as soon as the topoisomerase enzyme leaves the strands.

In this article, we will review how topoisomerase works, its application in lab assays, and the industrial areas involving the use of these enzymes.

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Structure of Topoisomerase

Topoisomerase is of two types: topo I and topo II. Both have different structures and functions. Have a look at what they are!

  • Type I Topoisomerase: Also known as DNA topoisomerase I.  This enzyme cuts the single strand of the DNA double helix and does not require ATP to perform the DNA cleavage. 

This is further divided into type IA, type IB (show homology to top I of human), and type IC. The only enzyme that introduces the positive supercoiling in DNA is a reverse gyrase enzyme, which is coupled with a helicase found in archaea and is a type IA topoisomerase.

  • Type II Topoisomerase: Also known as DNA topoisomerase II. It’s an ATP-dependent enzyme that cuts both the strands of the DNA substrate. In type II topoisomerases, ATPases are located either on the N-terminus (type IIA) or the B subunit (type IIB).

There are three types: type IIa (examples are DNA gyrase, topoisomerase IV, and human topoisomerase IIɑ and II𝛃), Type IIb, and Type IIC. These all enzymes catalyze negative supercoiling in DNA.

Figure: An illustration of different types of topoisomerase.

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.
Figure: An illustration of the working mechanism of type I topoisomerase.
  • 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.
Figure: An illustration of the working mechanism of type II topoisomerase.

Inhibition of Topoisomerase Enzyme

Some chemical compounds can inhibit the functional roles of topoisomerases in organisms. They commonly do it by hindering the DNA ligation step. Some examples of such chemicals are:

  • Protein inhibitors interfere with the DNA binding ability of the enzyme or stabilize the cleavage complex. Its examples are bacterial toxins MccB17, ParE, and YacG. 
  • Doxorubicin and its derivatives inhibit the functions of the human topoisomerases by stabilizing the cleavage complex.
  • The quinolone class of antibiotics inhibits topoisomerase activity by preventing the religation of broken DNA strands, which can cause mutation.
  • Mitoxantrone diacetate, which inhibits protein kinase activity, is also a type of potent topoisomerase II inhibitor.
Figure: A schematic diagram of topoisomerase inhibition.

What is Topoisomerase Used For?

Topoisomerase has a myriad of in vivo and in vitro applications in a range of metabolic processes, such as DNA replication, transcription, chromosome segregation, and recombination.  

DNA Replication

DNA replication is the process of synthesizing a new strand of DNA using the old one as a template. During the process, the topoisomerase enzyme help in introducing positive supercoiling ahead of the replication fork and catenation behind the fork.

In a closed genome replication process, such as a plasmid, the topoisomerase enzyme introduces negative supercoiling at the origin. This facilitates the melting of the strands and exposes them to start the replication process.

Transcription

Transcription is the synthesis of mRNA (or messenger RNA) by decoding information from the DNA strands. In this process, the topoisomerase enzyme helps introduce positive supercoiling ahead of the transcriptional complex and negative supercoiling behind it.

Double strand DNA breaks made by Topo IIβ and histone H-1 replacements are required to promote transcription. Furthermore, elements involved in DNA damage repair machinery are essential for gene expression and gene regulation. 

Recombination

Recombination is an exchange of genetic material between two chromosomes, leading to the formation of a different combination of genes. Topoisomerases are also integral components of the DNA repair complex. They are also involved in releasing knots formed during recombination. 

Chromosome Segregation

Topoisomerases are required to maintain a proper structure of the chromosome. It also helps in chromosome condensation, segregation, and cohesion. Furthermore, topoisomerase enzymes have essential roles in chromatin remodeling and centromere functions. 

What Industries Use Topoisomerase?

Topoisomerases have vast applications in all essential in vivo metabolic processes. Thus, the enzyme is commercially prepared and sold for use in in vitro workflows. Below are some industries that extensively use topoisomerases.

Pharmaceutical

In pharmaceuticals, topoisomerases are used as drug targets for antitumor or anti-cancer chemotherapy.  However, its inhibitors are used as antibiotics and in various anticancer therapeutics other than chemotherapy. 

Biochemistry

Topoisomerases have a range of applications in biochem labs. They help in studies ranging from in vitro replication and transcription to chromosome segregation and recombination in organisms. 

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Topoisomerases are enzymes that change DNA topology by breaking and relegating nucleic acid strands. The enzyme is also known as DNA topoisomerases as it has no function in RNA-related processes. 

Topoisomerases are of two types: type I, which creates single-strand breaks, and type II, which facilitates double-strand breaks in DNA substrate. The functions performed by the enzyme include catenation and decatenation, DNA supercoiling (increase or decrease supercoiling), and relaxing in metabolic processes like replication, transcription, and recombination.

Because of their extensive roles in a myriad of metabolic processes, topoisomerase enzymes are essential tools used in pharmaceutical and biochem labs. The result of these workflows depends greatly on the quality of the enzyme — in addition to high-throughput equipment.

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