lab meeting 01/28/13

Will, BPS practice talk

ISWI remodlers

  • can assemble arrays denov.  space nucleosomes out regularly — critical for silencing and folding
  • leading existing model: loop propagation
    • DNA binding domain scoops in 10 bp from entry side (feeds this into entry side).
  • 7 bp step, followed y two 3.5 bp steps.  (actually composed of 1 bp elementary steps).  (Exit side dynamics)
  • Entry side moves second
    • wait times are longer than exit side wait times.
    • restrict movement to less than 7 bp, exit side moves, but entry side does not.
  • ‘Spring-loaded model’
    • ATPase pumps in 7 bp of strain, which then leads to 3 bp steps of entry. Then reintroduce 3 bp of strain, get 3 bp of remodeling etc…
  • length sensing — study effect of linker length on remodeling.
  • pause phase decreases with linker length, translocation time doesn’t change
  • Regulatory domains of ISWI
    • AutoN, autoinhibitory model of ATP hydrolisis, competes with H4 tail recognition, makes enzyme H4 tail responsive (otherwise inhibited).
    • deleting DNA binding domain (HAND-SANT-SLIDE) does not substantially inhibit remodeling.
    • NegC inhibits coupling of hydorlysis and translocation.  is inhibited by HAND-SANT-SLIDE interaction with DNA.
  • Full model
    • AutoN inhibits ATP hydrolysis without histone tail to regulate
    • NegC is measuring distance by requring DNA interaction


  • is it important to say you label non-specifically and chose the subpopulation that has the cy3 you want?  (more info)
  • Introduce ISWI as a nucleosome spacer
  • published work is on how you move a nucleosome — nothing on spacing.  (linker length is motivated)
  • histone tails not motivated: — kinetics are slower in lysine-ac histones, this keeps ISWI from shuffling these regions around too much.
  • don’t return to published results after presenting unpublished results if possible.

Josh BPS practice talk

  • modified PCNA probe from fellow at CMU, better at strand invasion.   
  • Ultrabright probes.
  • individual fluorophores localized to single nm (Yildiz 2003).  all photons in one continuous burst, no switching.
  • ‘caged fluorphores’ — don’t switch back.
  • Cy3B-tublin + NaBH[4] (off).  add UV – 40% recovery.  16,000 photons per frame, 270,000 photons.  — localization uncertainty down to 3.7nm.
  • Alexa647 only 13%, Cy5.5 17%.  (1-2 million photons)
  • high intensity laser (more practical experiment lose photons (few 100,000).
  • direct labeled cy3B microtubules
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