Observation of RecBCD helicase/nuclease activity

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Observation of RecBCD helicase/nuclease activity. Bianco et al., Nature (2001) 409 : 374-378. Problems with using flow fields: a non-linear enzyme rate?. Bianco et al., Nature (2001) 409 : 374-378. UvrD unzips DNA without chewing it up. (conversion assay). - PowerPoint PPT Presentation

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  • Observation of RecBCD helicase/nuclease activityBianco et al., Nature (2001) 409: 374-378.

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

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

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

  • Unzipping, zipping and hybridization are observedVumax ~ Vzmax ~ 250 bp/sKM, ATP ~ 50 mMtu ~ tz ~ 1sDessinges et al., PNAS (2004) 101: 6439-6444

  • Measuring step-size by variance analysis

    mean distance travelled = NPDvariance of distance travelled = NP(1-P)D2Like 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 togetherFor UvrD, D ~ 6 +/- 1.5 bp

  • Single-molecule studies of DNA compactionPhages, Nucleosomes, Chromatin, Chromsomes,

  • Condensed states of DNA10s of microns of DNA length are stored invirus heads only tens of nm across. Meter lengths of DNA are stored in sperm heads and cell nuclei, tens of microns across.

  • Viral packaging of DNA(D.E. Smith et al. Nature 413 p.748, 2001)

  • Packaging in force-clamp modeF=5 pN; ~5.5 minutes to package f29 genome (~19kb)(D.E. Smith et al. Nature 413 p.748, 2001)

  • Packaging without feedbackForce increasesas bead gets pulledout of trap.(D.E. Smith et al. Nature 413 p.748, 2001)

  • External force vs. velocity dataNo feedback,low fillingA ~15 pN offset is observed between low and high-filling V vs. External Force data (D.E. Smith et al. Nature 413 p.748, 2001)

  • Internal pressure counteracts packing2x104 kBT forpackaging 20kbphage genome(similar to resultsFrom num.sim)(~ 6 MPa pressure)(D.E. Smith et al. Nature 413 p.748, 2001)

  • Force-velocity behaviorVmax ~ 100 bp/sD ~ 0.1 nmV ~ Vmax exp(-FD/kBT)(D.E. Smith et al. Nature 413 p.748, 2001)

  • Pause frequency, but not duration, is force- dependentThis suggests entry into a paused state is mechanosensitive, but not exiting from it.(D.E. Smith et al. Nature 413 p.748, 2001)

  • Two other DNA translocasesFtsKEcoRI 124

  • FtsK helps pump bacterial DNASaleh et al., EMBO J. (2004) 23, 24302439,

  • Forward translocation, dissociation, and reverse translocationVmax ~ 7 kbp/s!!Saleh et al., EMBO J. (2004) 23, 24302439,

  • Ejecting histones by applied force17 repeats of the sea urchin 5s positionning element = 17 nucleosomes at saturationBrower-Towland et al., PNAS (2001) 99 1960-1965

  • Single dissociation events detectedBrower-Towland et al., PNAS (2001) 99 1960-1965

  • Ejection is partially reversibleBrower-Towland et al., PNAS (2001) 99 1960-1965

  • Force-dependence of dissociationBrower-Towland et al., PNAS (2001) 99 1960-1965

  • dissociation of what?Brower-Towland et al., PNAS (2001) 99 1960-1965

  • DNA packaging by the X. laevis Condensin complexFrom Bazzett-Jones et al., Mol. Cell 9 1183-1190 (2002)

  • Proposed ModelFrom Bazzett-Jones et al., Mol. Cell 9 1183-1190 (2002)

  • DNA Compaction by X. laevis condensinFrom Strick et al., Curr. Bio. 14 874-880 (2004)

  • Direct observation of DNA compactionby mitotic condensina) Condensin + ATPb) Condensin - ATPc) Condensin + AMPPNPd) Interphasic condensin + ATPe) Condensin + ATP+ competitor DNA

  • Assembly of condensin onto supercoiled DNASkewed distributionswith peak at ~ 60 nm

  • Disassembly of DNA-bound condensin (yanking) 85 110 185Mean SD nSkewed distributionwith peak at ~ 30 nm

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