DNA topoisomerases in vivo Dr. Sevim 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 supercoiling; The linking number of DNA, a topological property, determines the degree of supercoiling; The linking number defines the number of times a strand of DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie in a plane; The linking number defines the number of times a strand of DNA winds in the right-handed direction around the helix axis when the axis is constrained to lie 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 = bp L=( ):10 = 44

Type I Topoisomerases Topo I of E. coli 1) acts to relax only negative supercoils 2) increases linking number by +1 increments Topo I of eukaryotes 1) acts to relax positive or negative supercoils 2) 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 Topoisomerases They 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. supercoils 2) Increases the linking number by increments of –2 3) Requires ATP Topo II of Eukaryotes 1) Relaxes only negatively supercoiled DNA 2) Increases the linking number by increments of +2 3) Requires ATP Negative Supercoiling DNA Gyrase Topo II Relaxation (-) supercoiled DNA relaxed DNA

Type II mechanism Cleavable Complex

Prokaryotes: Prokaryotes: Topo II of E. coli (DNA Gyrase) 1) Introduce neg. supercoils or relaxes pos. supercoils 2) Increases the # of neg. supercoils by increments of –2 3) Requires ATP relaxation supercoiling Reactions catalysed by topoisomerases Topo I of E. coli 1) acts to relax only negative supercoils 2) increases linking number by +1 increments

Reactions catalysed by topoisomerases Eukaryotes : Eukaryotes : Topo II of Eukaryotes 1) Relaxes only negatively supercoiled DNA 2) Increases the supercoiling by increments of +2 3) Requires ATP relaxation Topo I of eukaryotes 1) acts to relax positive or negative supercoils 2) changes linking number by –1 or +1 increments

knotting unknotting catenation decatenation Reactions catalysed by topoisomerases Knotting: 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 Replication DNA Replication Chromatin Condensation Segregation of Chromosomes during mitosis and meiosis Segregation of Chromosomes during mitosis and meiosis Transcription Recombination DNA Repair Functions of Topoisomerases

The role of topoisomerases in replication Initiation Initiation  Requirement for supercoiling  DnaA requires negative supercoiling to work Elongation Elongation  Requirement for relaxation of + supercoiling in front of replicatipon fork  Requirement for relaxation of excess (-) supercoiling behind replication fork Termination Termination  Removal of Catenanes (and precatenanes) Free rotation can not occur

Types of topoisomerases in replication Prokaryotes Prokaryotes Initiation Initiation  Gyrase: introduce negative supercoils at or near the oriC site in the DNA template Elongation  Gyrase : relax (+) supercoiling to introduce (-) sc Termination  Gyrase  Topo IV (a type II topo) remove catenanes

Types of topoisomerases in replication Eukaryotes Eukaryotes Initiation Initiation  Gyrase: introduce negative supercoils at or near the oriC site in the DNA template Elongation  Topo I: relax (+) supercoiling Termination  Topo II  : 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

Type 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. B acteria : T opoisomerase IV (topo IV) is the decatenase of DNA recombination intermediates. The f unction of topo IV is dependent on the level of DNA supercoiling. T he 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 Condensation Chromosome 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 Initiation Initiation  Promotion of helix opening by negative supercoiling Elongation Elongation  Requirement for topoisomerases to remove (+) supercoils ahead of the transcription machinary

Transcription - twin domains Free rotetion can not occur in vivo

Transcription - twin domains DNA Gyrase relaxes (+) supercoils Topo I relaxes excess (-) supercoils Eukaryotes : topo I removes both (+) & (-) supercoils

DNA topoisomerases as repair enzymes DNA 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 lesions 2) DNA recombination 3) regulation of DNA structure.

Topo II specific inhibitors