DNA topoisomerasesin vivo

DrDr.. Sevim IşıkSevim Işık

What is Supercoiling?

Positively supercoiled DNA is overwound

Relaxed DNA has no supercoils 10.4 bp

In addition to the helical coiling of the strands to form a double helix, the double stranded DNA molecule can also twist upon itself.

Supercoiling occurs in nearly all chromosomes (circular or linear)

Negatively supercoiled DNA is underwound (favors unwinding of the helix)DNA isolated from cells is always negatively supercoiled

The linking number of DNA, a topological property, determines the degree of The linking number of DNA, a topological property, determines the degree of supercoiling;supercoiling;

The linking number defines the number of times a strand ofThe linking number defines the number of times a strand of DNA winds in the DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie right-handed direction around the helix axis when the axis is constrained to lie in a plane;in a plane;

If both strands are covalently intact, the linking number cannot change; If both strands are covalently intact, the linking number cannot change;

Only topoisomerases can change the linking number.Only topoisomerases can change the linking number.

The Linking Number (L) of DNA

540 bp

L=540:10 = 54100 bp

L=(540-100):10 = 44

Type I Topoisomerases

Topo I of E. coli 1) acts to relax only negative supercoils2) increases linking number by +1 increments

Topo I of eukaryotes 1) acts to relax positive or negative supercoils2) changes linking number by –1 or +1 increments

They relax DNA by nicking then closing one strand of dublex. They cut one strand of the double helix, pass the other strand through, then rejoin the cut ends.

L = n L = n+1

All topoisomerases cleave DNA using a covalent Tyrosine-DNA intermediate

Type I mechanism

Because the relaxation (removal) of DNA supercoils by Topo I is energetically favorable, the reaction proceeds without an energy requirement.

Type II TopoisomerasesThey relax or underwind DNA by cutting both strands then sealing them. They change the linking number by increments of +2 or -2

Topo II of E. coli (DNA Gyrase) 1) Introduce negative supercoils or relaxes pos. supercoils2) Increases the linking number by increments of –23) Requires ATP

Topo II of Eukaryotes1) Relaxes only negatively supercoiled DNA2) Increases the linking number by increments of +23) Requires ATP

Negative Supercoiling

DNA Gyrase

Topo II

Relaxation

(-) supercoiled DNA

relaxed DNA

Type II mechanism

Cleavable Complex

Prokaryotes:Prokaryotes:

a

topo I

–ve sc DNA

DNA gyrase

+ve sc DNArelaxed DNA

DNA gyrase

Topo II of E. coli (DNA Gyrase) 1) Introduce neg. supercoils or relaxes pos. supercoils2) Increases the # of neg. supercoils by increments of –23) Requires ATP

relaxation relaxation

supercoiling

Reactions catalysed by topoisomerases

Topo I of E. coli 1) acts to relax only negative supercoils2) increases linking number by +1 increments

Reactions catalysed by topoisomerases

EukaryotesEukaryotes::

a

–ve sc DNA +ve sc DNArelaxed DNA

topoI

topo II

topoI

topo II

Topo II of Eukaryotes1) Relaxes only negatively supercoiled DNA2) Increases the supercoiling by increments of +23) Requires ATP

relaxation relaxation

Topo I of eukaryotes 1) acts to relax positive or negative supercoils2) changes linking number by –1 or +1 increments

knotting

unknotting

catenation

decatenation

Reactions catalysed by topoisomerasesKnotting: irreducible entanglement of a single DNA molecule

Catenation: the linking of two or more DNA molecules in which at least one strand of each dublex is in the form of a closed ring

If one strand is nicked, only then topo I catalyse catanation or decatanation

Type I or Type II topo

Type II topo

DNA ReplicationDNA Replication Chromatin Condensation Segregation of Chromosomes during mitosis and meiosisSegregation of Chromosomes during mitosis and meiosis Transcription Recombination DNA Repair

Functions of Topoisomerases

The role of topoisomerases in replication InitiationInitiation

Requirement for supercoilingRequirement for supercoiling DnaA requires negative supercoiling to workDnaA requires negative supercoiling to work

ElongationElongation Requirement for relaxation of Requirement for relaxation of + supercoiling+ supercoiling in front of in front of

replicatipon forkreplicatipon fork Requirement for relaxation of excess (-) supercoiling behind Requirement for relaxation of excess (-) supercoiling behind

replication forkreplication fork TerminationTermination

Removal of Removal of CatenaCatenanesnes (and precatena (and precatenanesnes))

Free rotation can not occur

Types of topoisomerases in replication ProkaryotesProkaryotes

InitiationInitiation Gyrase: introduce negative supercoils at or near the Gyrase: introduce negative supercoils at or near the

oriCoriC site in the DNA template site in the DNA template

ElongationElongation Gyrase : relax (+) supercoiling to introduce (-) sc Gyrase : relax (+) supercoiling to introduce (-) sc

TerminationTermination GyraseGyrase Topo IV (a type II topo)Topo IV (a type II topo)

remove catenanesremove catenanes

Types of topoisomerases in replication EukaryotesEukaryotes

InitiationInitiation Gyrase: introduce negative supercoils at or near the Gyrase: introduce negative supercoils at or near the

oriCoriC site in the DNA template site in the DNA template

ElongationElongation Topo I: relax (+) supercoilingTopo I: relax (+) supercoiling

TerminationTermination Topo IITopo II : remove catenanes : remove catenanes

Elongation of replication

negative supercoils

positive supercoils

precatenanes

leading strand

lagging strand

Precatenanes and (+) supercoils are formed in front of replication fork.

Elongation of replication

Topo I

Relaxation of (+) sc by topo I

Eukaryotes: Topo I relaxes positive supercoils ahead of replication fork

E. Coli DNA gyrase (adds neg. supercoils)

DNA gyrase

Prokaryotes DNA Gyrase remove positive supercoils that normally form ahead of the growing replication fork by adding negative supercoils

Elongation of replication

TypeType

Termination of replication

precatenanes

Topo II removes precatenanes at the end of replication

Prokaryotes : topo IV

Eukaryotes : topo II

Type II topoisomerases

The role of topoisomerases in recombination

DNA replication and recombination generate intertwined DNA intermediates that must be decatenated for chromosome segregation to occur.

Bacteria : Topoisomerase IV (topo IV) is the decatenase of DNA recombination intermediates. The function of topo IV is dependent on the level of DNA supercoiling. The role of gyrase in decatenation is to introduce negative supercoils into DNA, which makes better

substrates for topo IV.

Eukaryotes:Topo II decatenates the intertwined DNA intermediates. Topo I relaxes overwound DNA.

After DNA duplication, the chromosome pairs line up in a tetrad configuration .Adjacent chromosomes can exchange parts. Exchanging parts, simply mean that they exchange stretches of DNA.

Catenated (linked)

Replicated DNA molecules are separated (decatenated) by type II topoisomerases

Chromosome Segregation (decatanation)

topo IV

E. Coli : topo IV , Eukaryotes : topo II

Condensation cycle during replication

Decondensation

Replication

CondensationChromosome segregation

The role of topoisomerases in condensation Bacteria:Bacteria: free (-) supercoiling twists the dublex into a tightly interwound superhelix.free (-) supercoiling twists the dublex into a tightly interwound superhelix. DNA Gyrase introduce (-) supercoiling.DNA Gyrase introduce (-) supercoiling.

Eukaryotes:Eukaryotes: DNA is wrapped around histone octamers DNA is wrapped around histone octamers to form solenoidal (-) supercoils.to form solenoidal (-) supercoils.

Q: How Does Eukaryotic DNA Become Negatively Supecoiled?

Plectonemic supercoils

Solenoidal (Toroidal)supercoils

Q: What will happen if you remove the histone core?

A: DNA wrapping around histone cores leads to net negative supercoils!

Condensation

A: The solenoidal supercoil will adopt a plectonemic conformation

The role of topoisomerases in transcription

InitiationInitiation Promotion of helix opening by negative Promotion of helix opening by negative

supercoilingsupercoiling ElongationElongation

Requirement for topoisomerasesRequirement for topoisomerases to remove (+) to remove (+) supercoils ahead of the transcription machinarysupercoils ahead of the transcription machinary

Transcription - twin domains

aa

RNAP

mRNA

Ribosome

(a) Transcription elongation

(b) Schematic representation

(c) Formation of twin domains

(+)(–) (+) (+) (+)(–) (–) (–)

Free rotetion can not occur in vivo

Transcription - twin domains

a

DNA Gyrase relaxes (+) supercoils

Topo I relaxes excess (-) supercoils

Eukaryotes : topo I removes both (+) & (-) supercoils

DNA topoisomerases as repair enzymesDNA topoisomerases regulate the organization of DNA.

In addition, they modulate the cellular sensitivity toward a number of DNA damaging agents. Increased topoisomerase II activities contribute to the resistance of both nitrogen mustard-and cisplatin-resistant cells. Similarly, cells with decreased topoisomerase II levels show increased sensitivity to cisplatin, carmustine, mitomycin C and nitrogen mustard.

Topoisomerases may be involved in damage recognition and DNA repair at several different levels including:

1) the initial recognition of DNA lesions2) DNA recombination3) regulation of DNA structure.

 

Topo II specific inhibitors