Topoisomerase

Mr. Arvind Kumar

Topoisomerase

  • DNA is not just a straight ladder — in the cell, it’s supercoiled, twisted, and packed tightly.
  • Whenever DNA is opened (for replication, transcription, or repair), the double helix ahead of the opening gets overwound — like a phone cord twisting up.
  • This creates torsional stress that can stall or damage the process.

Topoisomerases are DNA “untangling” enzymes that:

  • Temporarily cut one or both DNA strands.

  • Allow controlled movement of DNA to remove twists, knots, or tangles.

  • Reseal the DNA without leaving gaps or errors.

Without them:

  • DNA would become so twisted that replication forks and RNA polymerases could not move forward.

  • Chromosomes would remain knotted or linked after replication and could not be separated during cell division.

Differences:

  • Type I: Cuts one strand; works in smaller steps; mostly ATP-independent.

  • Type II: Cuts both strands; can pass large segments through each other; requires ATP.

Topoisomerase Types

Topoisomerases are classified in two main ways:

Feature Type I Type II
Strands cut One Two
ATP use Usually none (except reverse gyrase) Yes
DNA movement Single-strand rotation or passage Double-strand passage
Functions Relieve supercoils Relieve supercoils, untangle DNA, introduce supercoils (in bacteria)

Type-I Topoisomerase

Type I topoisomerases cut one DNA strand and allow the DNA to rotate or pass a strand through before resealing.
Think of it like untying a rope by loosening just one side.

Structure

  • Generally, single protein chains (monomers).

  • Active site tyrosine attacks the DNA backbone to form a temporary phosphotyrosyl bond.

  • Has a DNA-binding groove shaped to hold DNA in place during cutting.

  • Subfamilies differ in domain arrangement and how they move DNA.

Subtypes of Type I

  1. Type IA

    • Cut one strand, pass the other strand through the gap.

    • Form 5′-phosphotyrosyl bonds.

    • Only relax negative supercoils.

    • Need single-stranded DNA regions to start.

    • Example: E. coli Topo I and Topo III.

  2. Type IB

    • Cut one strand and let it freely rotate to release supercoiling.

    • Form 3′-phosphotyrosyl bonds.

    • Can relax both positive and negative supercoils.

    • Example: human Topo I.

  3. Type IC

    • Found mostly in archaea (e.g., Topo V).

    • Mechanism like Type IB, but the structure is completely different.

Type IA Example: E. coli Topo I

  • Controls the negative supercoiling level in the cell.

  • Works with DNA gyrase to maintain proper DNA topology balance.

  • Topo III is more specialised for recombination intermediate resolution.

Mechanism of Type I Action 

For Type IB (free rotation model):

  1. The enzyme binds to the DNA helix.

  2. Active site tyrosine attacks the phosphodiester backbone, forming a 3′-phosphotyrosyl bond.

  3. The cut end of DNA swivels around the intact strand to relieve tension.

  4. The enzyme uses the free –OH group on DNA to attack the phosphotyrosyl bond, sealing the break.

For Type IA (strand passage model):

  1. Enzyme grips both single-stranded and double-stranded regions.

  2. Cuts one strand and opens a gate.

  3. Passes another strand through the gap.

  4. Reseals the DNA.

Functions

  • Relieve negative supercoils during transcription/replication.

  • Resolve single-stranded knots.

  • Prepare DNA for packaging into nucleosomes (in eukaryotes).

  • Help in recombination and DNA repair.

Type-II Topoisomerase

Type II enzymes cut both strands of DNA at the same time, pass another double-stranded segment through, and reseal.
Think of it like undoing a knot in a rope by opening it completely and pulling another rope section through.

Structure

  • Usually multimeric:

    • Bacteria: heterotetramers (A₂B₂).

    • Eukaryotes: homodimers.

  • Three major regions:

    1. ATPase domain (at the N-terminus) – binds/hydrolyses ATP to power conformational changes.

    2. DNA-cleavage/religation core – contains the active site tyrosines (one per strand).

    3. C-terminal domain – determines DNA preferences and cellular roles.

Subtypes

  • Type IIA: most common in bacteria and eukaryotes.

    • Examples: DNA gyrase, Topo IV, eukaryotic Topo IIα, Topo IIβ.

  • Type IIB: found in archaea and plants.

    • Example: Topo VI – related to the Spo11 protein in meiosis (causes programmed DSBs).

Mechanism of Type II Action

  1. Bind the G-segment (the one to be cut) in the cleavage core.

  2. Capture T-segment in the ATPase domain.

  3. Hydrolyse ATP to close the ATPase gate, trapping the T-segment.

  4. Cut both strands of G-segment, hold ends covalently via active site tyrosines.

  5. Pass the T-segment through the break.

  6. Reseal the G-segment and release the T-segment.

  7. Hydrolyse ATP to reset the enzyme.

Functions

  • Decatenate (unlink) replicated chromosomes.

  • Relax both positive and negative supercoils.

  • In bacteria, gyrase introduces negative supercoils to compact DNA.

  • In eukaryotes, Topo IIα condenses chromosomes during mitosis.

Topoisomerase Inhibition

Topo I Inhibitors (mainly cancer drugs)

  • Camptothecin (natural alkaloid from Camptotheca acuminata).

  • Topotecan – used for ovarian and small-cell lung cancer.

  • Irinotecan – used for colorectal cancer.

  • Mechanism: stabilise the covalent Topo I–DNA complex → replication collision → DNA double-strand breaks.

Topo II Inhibitors (cancer drugs)

  • Etoposide and Teniposide – block resealing step.

  • Doxorubicin, Mitoxantrone – intercalate into DNA and trap Topo II.

  • Side effects: bone marrow suppression, possible secondary leukaemias (due to DNA damage in healthy cells).

Bacterial Topo II Inhibitors

  • Fluoroquinolones (ciprofloxacin, levofloxacin) – target gyrase and Topo IV.

  • Cause DNA double-strand breaks in bacteria → cell death.

  • Widely used but resistance is growing.

 

Clinical Significance

  • Essential for cell survival → attractive drug targets.

  • Cancer therapy: Topo poisons kill rapidly dividing cells.

  • Antibiotics: Target bacterial topoisomerases without affecting human ones.

  • Resistance mechanisms: mutations in binding sites, drug efflux, protective proteins.

 

Topoisomerase vs Helicase

  • Helicase: unwinds DNA strands using ATP.

  • Topoisomerase: relieves twisting pressure caused by helicase and other processes.

  • Without topoisomerase, helicase would stall due to supercoiling ahead of the fork.

Feature Topoisomerase Helicase
Main job Removes supercoils, untangles DNA, and separates linked DNA circles Unwinds the double helix into two single strands
Action Cuts DNA (one or both strands), moves DNA to relieve twist, then reseals Breaks hydrogen bonds between bases to separate strands
DNA strands cut? Yes – Type I cuts one, Type II cuts both No cutting – just strand separation
ATP usage Type I: usually no (except reverse gyrase); Type II: yes Yes – requires ATP hydrolysis to move along DNA
When it works Ahead of replication forks, transcription bubbles, and during chromosome segregation At replication forks during DNA synthesis
Directionality Not strongly directional – acts where needed Moves in a specific 5′→3′ or 3′→5′ direction along DNA
Effect on DNA Changes DNA topology (supercoiling, knotting, catenation) Produces single-stranded templates for replication or repair
Coordination Works with helicase – prevents overwinding caused by strand separation Works with topoisomerase – creates the supercoils that need to be relieved

Topoisomerase vs Gyrase

  • Gyrase is a bacterial Type II topoisomerase that can introduce negative supercoils.

  • Helps bacteria compact DNA and make it ready for transcription.

  • Targeted by antibiotics like fluoroquinolones.

  • No equivalent enzyme in humans → good selective drug target.

Feature Topoisomerase (General) DNA Gyrase
Definition Enzymes that change DNA topology by cutting and rejoining DNA strands to remove or add supercoils A Type II bacterial topoisomerase that can introduce negative supercoils into DNA
Occurrence Found in all organisms (bacteria, archaea, eukaryotes) Found only in bacteria and some archaea
Types Type I (cuts 1 strand) and Type II (cuts 2 strands) Type II only (heterotetramer: GyrA₂GyrB₂)
Supercoil activity Mostly relaxes supercoils; some (like reverse gyrase) add positive supercoils Introduces negative supercoils and can also relax supercoils
ATP requirement Type I: usually no; Type II: yes Yes – requires ATP hydrolysis
Functions Relieve torsional stress, untangle DNA, separate linked chromosomes Maintain negative supercoiling in bacterial chromosomes; assist in replication, transcription, recombination
Drug targets Anti-cancer (e.g., etoposide, camptothecin) and antibacterial (fluoroquinolones target bacterial topoisomerases) Primary target of many fluoroquinolone antibiotics (e.g., ciprofloxacin)
Presence in humans Yes – humans have several topoisomerases No – gyrase is absent in humans, making it a good selective antibacterial target