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DNA Topoisomerases

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

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DNA Topoisomerases

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  1. DNA Topoisomerases

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

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

  4. Topological issues in DNA replication

  5. 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? Supercoiling and transcription

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

  7. Eukaryotic Topoisomerases

  8. Mechanisms of Type II Topoisomerases

  9. 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) Therapeutic Implications

  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 mm) ATP • Two supercoils relaxed per catalytic turnover • Tcycle displays single-exponential statistics

  12. Processive activity at higher [ATP] Magnet rotation applied Topo II activity • Trelax << Twait 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 Km = 270 mM ATP Vsat = 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 High processivity (commonly 40, up to 80 reported) V0 = 2.7 cycles/s, D = 1.9 nm  Enzyme rate is not torque-sensitive Charvin et al., PNAS (2003) 100: 115-120

  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 Processive Distributive

  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. (lt)n ___ exp(-lt) P(n) = n! Measuring step-size by variance analysis Random • X(t) is the recorded position of the system • Record many (long) traces and average them together • mean = <X> = NPD • variance = < (X - < X >)2 > = NP(1-P)D2

  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. variance = D mean Measuring step-size by variance analysis Like a random walk: N steps with a probability P (small) of moving forward a distance D Repeat the walk a large number of times and average the results together mean distance travelled = NPD variance of distance travelled = NP(1-P)D2

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