Topoisomerase enzymes

1. TOPOISOMERASES
S. Aishwarya
2nd M.Sc. Biochemistry

2. Definition
■ A class of enzymes that alter the supercoiling of double-stranded DNA. (In
supercoiling the DNA molecule coils up like a telephone cord, which shortens
the molecule.) The topoisomerases act by transiently cutting one or both strands
of the DNA.
■ The regulation of DNA supercoiling is essential to DNA transcription and
replication, when the DNA helix must unwind to permit the proper function of the
enzymatic machinery involved in these processes.
■ Topoisomerases serve to maintain both the transcription and replication of DNA.

3. Continued..
■ Topoisomerases change the Lk of a circularly wound double stranded
DNA.(Lk- total number of times one strand of DNA helix is linked with the
other in a covalently closed circular molecule)
■ Change in Lk causes change in Wr.(number of times the DNA helix is
coiled about itself in 3d space)
■ Variation in Wr subsequently changes the state of compaction of DNA
molecule.
■ This is advantageous because it permits the DNA to be transiently and
locally melted to permit the enzymes of DNA replication and transcription
to copy and synthesize new DNA or RNA

4. Types
Type 1 topoisomerases
■ These enzymes remove
supercoils by breaking only one of
the two strands of the DNA.
■ As a result enzyme change the LK
by 1 each time.
Type 2 topoisomerases
■ In this both the phosphodiester
backbone chains are broken
simultaneously.
■ LK changes by 2.

5. Type 1 topoisomerases
■ Best characterised is E.coli.
STRUCTURE
■ Enzyme is 864 a.a long
■ Monomeric
■ Encoded by topA gene

6. Mechanism of action
■ Mechanism of catalysis involves formation of covalent residue between tyrosine
residue and the Phosphodiester backbone.
■ Nucleophilic attack from hydroxyl group of tyrosine to a phosphorus atom
creates a phosphodiester link between the enzyme and the DNA generates a
free 5’ hydroxyl group.
■ The other strand is held in place by binding non covalently to a domain of the
enzyme.
■ The strand is resealed.
■ Thus one supercoil is removed.
■ Topo 1 from E.coli acts only on negative supercoil.
■ Eukaryotic Topo 1 can remove both negative and positive supercoil.

9. Topoisomerase 2
■ Best characterised is E.coli Topo 2 known as DNA gyrase.
STRUCTURE
■ DNA gyrase is tetrameric protein with two A subunits (875a.a) and two B
subunits (804a.a)
■ Depending on DNA substrates they can change positive supercoils to negative
or increase the number of negative supercoil by 2.
■ Topo 2 catylse carnation and decatenation i.e, linking and unlinking two DNA
template
■ The enzyme also introduces negative or positive supercoils at or near the ori C
site.
■ It also removes positive supercoils formed ahead of replication.

10. Mechanism of action
■ Hydrolysis of ATP by ATPase activity of gyrase causes conformational change.
■ Initially enzyme binds to one part of DNA strand – G segment inducing
conformational changes in all the a and b subunits.
■ After binding the ATP and another strand – T segment , a series of reactions
occur in which G segment is cut by a and a’ domains of the enzyme and the
ends of the G DNA become covalently linked to tyrosine residues in these
domains.
■ Simultaneously, the ATP binding domains move towards each other, transporting
the T segment through the break.
■ The cut G segment is resealed and T segment is released by conformational
change that separates a and a’ domains then reforms , a reaction that requires
ATP hydrolysis and regenerates the starting state.
■ Alternatively the enzyme can proceed another cycle.

12. Inhibitors of topoisomerases
■ They are essential enzymes.
■ Mutation of any of the gene coding for topoisomerase are usually lethal.
■ Therefore they are targets for antibiotics.
Bacteria can be killed by novobiocin or nalidix acid. Both of these inhibit DNA
gyrase.
Novobiocin blocks ATP binding and nalidixic acid blocks the breakage and
rewinding mechanism. These antibiotics do not inhibit eukaryotic topoisomerases
and are useful in eradicating bacterial infections.
■ Eukaryotic topoisomerase inhibitors such as doxorubicin and etoposide are
used as chemotherapeutic agents.

13. Thank you