​Topoisomerase​ – Structure, Inhibitors & Role in DNA Replication

Topoisomerases are nucleases that break the phosphodiester bonds in DNA reversibly. These enzymes are important in relieving the positive supercoiling that arises from DNA unwinding mediated by helicases during replication. The first topoisomerase was discovered by James Wang in 1971 from Escherichia coli. There are diverse types of Topoisomerases known to date. All these Topoisomerases can be classified into two types- Type I Topoisomerase and Type II Topoisomerase based on their ability to cleave one strand or two strands of DNA respectively.

Define Topoisomerase

Topoisomerases are nuclear enzyme that are responsible for relieving the supercoiling of DNA and maintaining DNA topology during several cellular processes like replication, transcription, and chromosome segregation.

What is Topoisomerase?

Topoisomerase is a nuclear enzyme that relieves positive supercoiling of DNA during different cellular processes like replication, transcription, chromosome segregation, etc. It does so by breaking the phosphodiester bonds of DNA reversibly. There are several types of DNA Topoisomerases in eukaryotes and prokaryotes. Some of them could cleave only one strand of the DNA and some of them could cleave both the strands. Depending on the number of strands they can cleave they are classified as Type I Topoisomerase and Type II Topoisomerase. Type I can cleave only one strand whereas Type II can cleave both strands.

Types of Topoisomerase

Primarily there are two types of Topoisomerases: Type I Topoisomerases and Type II Topoisomerases. Both these types are differentiated based on their function, structure, and the mechanism by which they cleave DNA. Both these types are discussed further.

Type I Topoisomerase

Type I Topoisomerases are involved in relieving torsional stress and managing the supercoiling of DNA by introducing single-strand breaks and allowing the DNA to pass through the gap or rotate around the intact strand.

• There are two main subtypes within Type I Topoisomerases: Type IA and Type IB.
• Type IA Topoisomerases covalently bind 5′-phosphate during DNA hydrolysis and effect Topological changes in DNA through a ‘strand pass’ mechanism in which it cleaves one strand of the dsDNA and allows the other strand to pass through the gap followed by resealing.
• On the other hand, Type IB Topoisomerase binds to 3′-phosphate and relieves supercoiling by nicking a single strand of DNA and allowing other strand end to rotate with respect to other around the intact phosphodiester bond on the oppposite strand.
• Type IA Topoisomerases can be grouped into three classes- eubacterial Topo IA, eubacterial and eukaryotic Topo III and eubacterial and archaeal reverse gyrase whereas Type IB Topoisomerase is represented by Topo IB only.

Type II Topoisomerase

Type II Topoisomerases regulate the DNA topology by introducing double-strand breaks in DNA molecules. They are involved in processes such as DNA replication, transcription, and chromosome segregation during cell division.

• There are two main subtypes within Type II Topoisomerases: Type IIA and Type IIB.
• Both Type IIA and Type IIB Topoisomerase uses duplex strand passage mechanism and have almost same core but there overall tertiary structure differs.
• Type IIA Topoisomerases are found in all cellular organisms, as well as in viruses. They are divided into three classes eukaryotic Topo II, bacterial topo IV, and bacterial and archaeal DNA gyrase.
• Type IIB Topoisomerases include topo IV from plant and homologues of Spo 11 from yeast.

Functions of Topoisomerase

The ability of Topoisomerases to break phosphodiester bonds reversible makes them crucial components in many cellular processes like replication, transcription, seggregation, etc. Primary functions of Topoisomerases are as follows:

• Relieving Torsional Stress: DNA Topoisomerases help alleviate torsional stress and strain in DNA that arises during various cellular processes, such as DNA replication, transcription, and recombination. Thereby prevent the overwinding (positive supercoiling) or underwinding (negative supercoiling) of DNA strands.
• Unwinding and Untangling of DNA: During processes like DNA replication, the DNA double helix needs to be unwound to expose the template strands for copying. Topoisomerases, especially Type II, untangle and unwind the DNA by introducing transient double-strand breaks, allowing the passage of another DNA segment through the break.
• Facilitating DNA replication, transcription, and seggregation: Topisomerases relieve over winding of DNA thus help in smooth execution of any of the process that involve DNA unwinding by creating a smooth path for enzymes involved. During segregation they uncoil the intertwined DNA and jelps in proper segregation.
• Therapeutic targets: The Topisomerases serve as targets of some drugs that are use in differen types of cancer treatment. These drugs interrupt with Topoisomerase activity and kill rapidly dividing cells.

Topoisomerase Inhibitors and Clinical Significance

Topoisomerase inhibitors are drugs that interfere with the activity of Topoisomerases, crucial enzymes involved in DNA topology regulation. They are widely used in cancer chemotherapy due to their ability to disrupt DNA replication and repair, leading to cell death. There are two main types: Topoisomerase I inhibitors and Topoisomerase II inhibitors. However, some drugs, like the fluoroquinolone antibiotics (e.g., ciprofloxacin), act as dual inhibitors of both Topoisomerase I and Topoisomerase II.

Topoisomerase I inhibitors

• Topoisomerase I inhibitors are together called Campothecins.
• This class of drugs includes camptothecin, topotecan, and irinotecan.
• Camptothecins stabilize the covalent complex formed between Topoisomerase I and DNA, preventing religation of the cleaved DNA strand. This results in the accumulation of DNA breaks and inhibition of DNA replication.

Topoisomerase II inhibitors

• Anthrocyanines, Anthracycline, Podophyllotoxin Derivatives, and Amsacrine are common classes of drugs that function as Topoisomerase II inhibitors.
• All these drugs stabilize the cleavable complex formed by Topoisomerase II with DNA, preventing the enzyme from completing the religation step.
• This leads to the accumulation of double-strand breaks and inhibition of DNA replication and transcription finally resulting in cell death.

Although these inhibitors are effective in targeting rapidly dividing cancer cells, they can also affect normal cells, leading to side effects. Researchers continue to explore novel Topoisomerase inhibitors with improved selectivity and reduced toxicity for enhanced cancer therapy.

Difference Between Topoisomerase and Helicase

Topoisomerase and helicase are important nucleases that are infolved in uncoiling of the DNA helix however their functions are quite different. The differences are listed below.

Criteria	Topisomerase	Helicase
Function	Regulate DNA topology by breaking and rejoining strands.	Unwind the DNA or double helix by disrupting hydrogen bonds.
Target and Action	Act on entire DNA molecule; introduce transient breaks by breaking the phosphodiester bond.	Specifically target and unwind DNA at replication fork or other sites by breaking the hydrogen bond.
Location in DNA Replication	Act at various points to manage supercoiling.	Play a key role at the replication fork by unwinding DNA strands.
Energy Source	Energy often derived from breaking and rejoining DNA strands.	Energy comes from ATP hydrolysis.
Types	Type I Topoisomerase I and Type II Topoisomerase.	DNA helicase and RNA helicase.

Difference Between Topoisomerase and Gyrase

Gyrase is a type of Topoisomerase but it has some typical characters that distinguishes it from other Topisomerases in the group.

Criteria	Topoisomerase	Gyrase
Classification	These include a large family of nucleases that can cause DNA single strand break or double strand break.	Gyrase is a type of Type II Topoisomerase.
Function	Regulate DNA topology by breaking and rejoining strands.	Introduce negative supercoils into DNA.
Occurence	Present in prokaryotes as well as eukaryotes.	Commonly present in prokaryotes.
ATP requirement	They may or may not use ATP .	They need ATP.

Conclusion – Topoisomerase

Topoisomerases are vital enzymes in cellular processes, they relieve DNA supercoiling and ensure genomic stability.

1. Topoisomerase I induces transient single-strand breaks, relieving tension by allowing rotational movement of the DNA strands.
2. Topoisomerase II, on the other hand, introduces reversible double-strand breaks, enabling the passage of another DNA segment through and subsequent resealing.
3. These enzymatic actions are crucial for DNA replication, transcription, and chromosome segregation during cell division, highlighting their essential role in maintaining the integrity and proper functioning of the genetic material in living cells.

FAQs – Topoisomerase

What is the Function of Topoisomerase?

Topoisomerases play a crucial role by relieve torsional stress and prevent overwinding or underwinding of the DNA helix by creating transient breaks in one or both DNA strands.

How does Topisomerase differ from Helicase?

Topoisomerases regulate DNA topology by introducing transient breaks to relieve torsional stress, while helicases unwind the DNA double helix by disrupting hydrogen bonds.

Does Topoisomerase break Phosphodiester Bonds?

Yes, Topoisomerases break and then rejoin the phosphodiester bonds in DNA strands.

Where is Topoisomerase Used in Medicine?

Topoisomerases are widely used for cancer treatment as they inhibit Topoisomerase activity in religation step and ultimately result in cell death.

What are the Examples of Topoisomerases?

Bacterial and archaeal Gyrase is an example of Type II Topoisomerase. This enzyme introduces negative supercoiling of DNA helix and helps in the advancement of replication fork.

What is the Difference between Topoisomerase I and II?

Topoisomerase I resolves DNA supercoiling by creating a transient single-strand break, while Topoisomerase II introduces a double-strand break and passes another DNA segment through it, altering DNA topology.

What is the Mechanism of Action of Topoisomerase?

All topoisomerases target the phosphodiester backbone of DNA to produce a strand break.