DNA topoisomerases in vivo

1. DNA topoisomerasesin vivo Dr.Sevim Işık

2. What is Supercoiling? 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) Relaxed DNA has no supercoils10.4 bp Negatively supercoiled DNA is underwound (favors unwinding of the helix) DNA isolated from cells is always negatively supercoiled Positively supercoiled DNA is overwound

3. The Linking Number (L) of DNA The linking number of DNA, a topological property, determines the degree of supercoiling; The linking number defines the number of times a strand ofDNA 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; Only topoisomerases can change the linking number. 540 bp 100 bp L=540:10 = 54 L=(540-100):10 = 44

4. Type I Topoisomerases 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 Topo I of eukaryotes 1) acts to relax positive or negative supercoils 2) changes linking number by –1 or +1 increments Topo I of E. coli 1) acts to relax only negative supercoils 2) increases linking number by +1 increments

5. Type I mechanism All topoisomerases cleave DNA using a covalent Tyrosine-DNA intermediate Because the relaxation (removal) of DNA supercoils by Topo I is energetically favorable, the reaction proceeds without an energy requirement.

6. 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 relaxed DNA DNA Gyrase Relaxation Negative Supercoiling Topo II (-) supercoiled DNA 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

7. Type II mechanism Cleavable Complex

8. Reactions catalysed by topoisomerases • Prokaryotes: relaxation relaxation supercoiling Topo I of E. coli 1) acts to relax only negative supercoils 2) increases linking number by +1 increments 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

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

10. Reactions catalysed by topoisomerases Knotting:irreducible entanglement of a single DNA molecule Type I or Type II topo knotting unknotting 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 Type IItopo catenation decatenation If one strand is nicked, only then topo I catalyse catanation or decatanation

11. Functions of Topoisomerases • DNA Replication • Chromatin Condensation • Segregation of Chromosomes during mitosis and meiosis • Transcription • Recombination • DNA Repair

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

13. Types of topoisomerases in replication • Prokaryotes 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

14. Types of topoisomerases in replication • Eukaryotes 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

15. Elongation of replication Precatenanes and (+) supercoils are formed in front of replication fork. leading strand lagging strand precatenanes positive supercoils negative supercoils

16. Elongation of replication Eukaryotes: Topo I relaxes positive supercoils ahead of replication fork Topo I Relaxation of (+) sc by topo I

17. Elongation of replication Prokaryotes DNA Gyrase remove positive supercoils that normally form ahead of the growing replication fork by adding negative supercoils DNA gyrase E. Coli  DNA gyrase (adds neg. supercoils)

18. Termination of replication Topo II removes precatenanes at the end of replication Type precatenanes Type II topoisomerases Prokaryotes : topo IV Eukaryotes : topo II

19. The role of topoisomerases in recombination 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. DNA replication and recombination generate intertwined DNA intermediates that must be decatenated for chromosome segregationto occur. Bacteria : Topoisomerase IV (topo IV) isthe decatenase of DNA recombination intermediates. The function of topo IV is dependent on the level of DNAsupercoiling. The role of gyrase in decatenationis to introduce negative supercoils into DNA, which makes bettersubstrates for topo IV. Eukaryotes: Topo II decatenates the intertwined DNA intermediates. Topo I relaxes overwound DNA.

20. Chromosome Segregation (decatanation) Replicated DNA molecules are separated (decatenated) by type II topoisomerases Catenated (linked) topo IV E. Coli : topo IV , Eukaryotes : topo II

21. Condensation cycle during replication Decondensation Replication Chromosome segregation Condensation

22. The role of topoisomerases in condensation • Bacteria: free (-) supercoiling twists the dublex into a tightly interwound superhelix. DNA Gyrase introduce (-) supercoiling. • Eukaryotes: DNA is wrapped around histone octamers to form solenoidal (-) supercoils.

23. Condensation Q: What will happen if you remove the histone core? Plectonemic supercoils Solenoidal (Toroidal) supercoils A: The solenoidal supercoil will adopt a plectonemic conformation Q: How Does Eukaryotic DNA Become Negatively Supecoiled? A: DNA wrapping around histone cores leads to net negative supercoils!

24. The role of topoisomerases in transcription • Initiation • Promotion of helix opening by negative supercoiling • Elongation • Requirement for topoisomerases to remove (+) supercoils ahead of the transcription machinary

25. Transcription - twin domains Free rotetion can not occur in vivo

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

27. 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 lesions2) DNA recombination3) regulation of DNA structure.

28. Topo II specific inhibitors