Will, BPS practice talk
- 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- don’t return to published results after presenting unpublished results if possible.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 (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