1 / 29

Ku-DNA Interaction

Ku-DNA Interaction . By Enaefe Ziregbe. Fluorescence Anisotropy: how the thermodynamics of Ku-DNA interaction were characterized. DNA non-homologous end joining starts with the binding of Ku heterodimers to double strand breaks. Flourescence Anisotropy. What is it?

fadhila
Download Presentation

Ku-DNA Interaction

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  1. Ku-DNA Interaction By Enaefe Ziregbe

  2. Fluorescence Anisotropy: how the thermodynamics of Ku-DNA interaction were characterized. • DNA non-homologous end joining starts with the binding of Ku heterodimers to double strand breaks.

  3. Flourescence Anisotropy • What is it? -light emitted by the fluoropore has unequal intensities along different axes of polarization.

  4. Ku-DNA binding • Kd for Ku-DNA binding site of the proper length (>20bp) ranges from 22 to 29nM at 300mM NaCl. • DNA homologous end joining is the predominant pathway of DNA repair in multicellular eukaryotes.

  5. Proteins involved in NHEJ • Ku 70 • Ku 80 • DNA-PKcs • Xrcc4 • DNA ligase IV • Artemis

  6. DNA Ligase IV and Xrcc4 Form a tight complex

  7. Ku 80, Ku 70 and DNA-PKcs • Constitutes a complex known as DNA protein kinase.

  8. Ku heterodimer. Credit: Wikepedia

  9. http://www.youtube.com/watch?v=xS6bJMcOrAk

  10. Structure • Two subunits are highly interlocked and form a ring to with a large narrow base and a narrow handle that threads the DNA in an energy-dependent manner.

  11. Ku heterodimer • How is it formed? Formed by the Ku70 and Ku80 subunits. • First molecule to bind DNA ends generated by double strand break invivo. • After Ku molecules bind to DNA ends can slide freely on dsDNA chain and their presence does not favor or impair binding of additional heterodimers to the DNA break.

  12. Ma and Lieber -proposed that Ku binds a 45bp DNA duplex in a cooperative fashion but the cooperativity is lost when using longer duplexes.

  13. How true is this? - Six different DNA duplexes of various lengths were labeled with a fluorescent probe and measured the binding isotherms by following the change in the anisotropy signal upon formation of the DNA complex.

  14. S20 (o)and S25(.)

  15. Stoichiometric Determination for s42 and s50 duplexes

  16. s75

  17. This approach allowed accurate determinations of the dissociation constants under equilibrium conditions. • The binding isotherms obtained were analyzed using both a conventional non-overlapping method and a more overlapping binding site model.

  18. Effect of Salt on Ku-DNA • Quantitative studies on salt concentration on Ku-DNA were performed to obtain information on the number of cations and anions released upon formation of the complex as well as an estimate of the electrostatic component of the binding free energy.

  19. Sources • Fluorescence Anisotropy Studies on the Ku-DNA interaction by Daniele Arosio, Silvia Costantini, Yong Kong, and Alessandro Vindigni • http://www.jbc.org/content/279/41/42826.full.pdf

  20. QUESTIONS??

More Related