1. DNA Topoisomerases

2. DNA Supercoiling in vivo In most organisms, DNA is negatively supercoiled (  ~ -0.06) Supercoiling is involved in initiation of transcription, replication, repair & recombination Actively regulated by topoisomerases, ubiquitous and essential family of proteins

3. Chromosomes: the ultimate Gordian knot? EM by U. Laemmli

4. Topological issues in DNA replication

5. Supercoiling and transcription In bacteria, gyrase helps maintain negative supercoiling. This can help drive transcription in many genes (although gyrase is, itself, downregulated by negative supercoiling). Mutations in gyrase are compensated by mutations in topo I to prevent it from removing negative supercoiling. Positive supercoils ahead of RNAP, negative supercoils behind?

6. Bacterial Topoisomerases VIRAL TOPOISOMERASES: vaccinia (smallpox), phage T4 Topo II

7. Eukaryotic Topoisomerases

8. Mechanisms of Type II Topoisomerases

9. Therapeutic Implications Gyrase is a good target for antibacterial quinolones (ciproflaxin). DNA Breakages are toxic… Reversed by tyrosyl-DNA phosphodiesterases (3’ topo Ib breaks)… How are tdp proteins and other break-repairing proteins (involved in recombinational repair) involved in resistance to chimiotherapeutic agents? Topoisomerase II poisons are used in chemotherapy (daunorubicin, doxorubicin, etoposide) as well as Topo I poisons (topotecan)

10. How to detect topoisomerase activity in a single-molecule assay  is calibrated by measuring the change in DNA extension observed for a unit rotation of the bead

11. Single turnovers observed at low (10  m) ATP Two supercoils relaxed per catalytic turnover T cycle displays single-exponential statistics

12. Processive activity at higher [ATP] Topo II activity Magnet rotation applied T relax << T wait  single molecule bursts Processivity on the order of ten cycles

13. DNA crossovers are the substrate of topo II

14. Eurkaryotic Topo II does not distinguish (+) and (-) sc

15. [ATP] and force-dependence of strand passage K m = 270  M ATP V sat = 3 cycles/sec Rate-limiting step coupled to ~1nm motion against the applied force

16. How do we know this is not torque-related? Charvin et al., PNAS (2003) 100: 115-120

17. Decatenation Experiments show similar Kcat V 0 = 2.7 cycles/s,  = 1.9 nm High processivity (commonly 40, up to 80 reported) Charvin et al., PNAS (2003) 100: 115-120  Enzyme rate is not torque-sensitive

18. Model: closure of the DNA gap is rate-limiting

19. Principle of “clamping” experiment

20. Topo II binds to DNA crossovers

21. Detection of individual clamping events (DNA is pre-twisted to the threshold of the buckling transition)

22. Clamping lifetimes: with Magnesium

23. Bacterial Topo IV distinguishes (+) and (-) sc Distributive Processive

24. Again: is torque driving this effect?? Use braided DNA molecules to measure effect of topology without torque Charvin et al., PNAS (2003) 100: 115-120

25. Force-response of bacterial topo IV L-braids (topologically equivalent to + supercoils) are removed more quickly than R-braids (~ – supercoils) Final R-braid crossover very hard to remove (as opposed to final L-braid crossover. Topo IV cycle less mechanosensitive than topo II cycle. At the same time, characteristic length-scale for work against force at rate-limiting mechanosensitive step involves displacement against force over a distance of ~10 nm (5x greater than topo II) Charvin et al., PNAS (2003) 100: 115-120

26. Topo IV can remove R-braids if they supercoil (thus forming L-crossovers) Charvin et al., PNAS (2003) 100: 115-120

27. Type I Topoisomerases: a comparison Topo Ia Topo Ib

28. Measuring step-size by variance analysis 1.X(t) is the recorded position of the system 2.Record many (long) traces and average them together mean =  X  = NP  variance =  X -  X    = NP(1-P)  2 ( t) n n! ___ exp(- t) P(n) = Random

29. Observation of RecBCD helicase/nuclease activity Bianco et al., Nature (2001) 409: 374-378.

30. Problems with using flow fields a non-linear enzyme rate? Bianco et al., Nature (2001) 409: 374-378.

31. UvrD unzips DNA without chewing it up (conversion assay) Dessinges et al., PNAS (2004), 101: 6439--6444

32. At low force DNA hybridization is a problem Dessinges et al., PNAS (2004), 101: 6439--6444

33. Unzipping, zipping and hybridization are observed Dessinges et al., PNAS (2004), 101: 6439--6444

34. Measuring step-size by variance analysis mean distance travelled = NP  variance of distance travelled = NP(1-P)  2 Like a random walk: N steps with a probability P (small) of moving forward a distance  Repeat the walk a large number of times and average the results together mean variance = 