The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity

Hart, D., Speight, R.E., Cooper, M.A., Sutherland, J.D., & Blackburn, J.M. (1999) The salt dependence of DNA recognition by NF-kappaB p50 : a detailed kinetic analysis of the effects on affinityand specificity. Nucleic Acids Research, 27(4), pp. 1063-1069.

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The binding kinetics of NF-kappaB p50 to the Ig-kappaB site and to a DNA duplex with no specific binding site were determined under varying conditions of potassium chloride concentration using a surface plasmonresonance biosensor. Association and dissociation rate constants were measured enabling calculation of the dissociation constants. Under previously established high affinity buffer conditions, the k a for both sequences was in the order of 10(7) M-1s-1whilst the k d values varied 600-fold in a sequence-dependent manner between 10(-1) and 10(-4 )s-1, suggesting that the selectivity of p50 for different sequences is mediated primarily through sequence-dependent dissociation rates. The calculated K D value for the Ig-kappaB sequence was 16 pM, whilst the K D for the non-specific sequence was 9.9 nM. As the ionic strength increased to levels which are closer to that of the cellular environment, the binding of p50 to the non-specific sequence was abolished whilst the specific affinity dropped to nanomolar levels. From these results, a mechanism is proposed in which p50 binds specific sequences with high affinity whilst binding non-specific sequences weakly enough to allow efficient searching of the DNA.

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ID Code: 70423
Item Type: Journal Article
Refereed: Yes
Additional Information: Articles free to read on journal website
DOI: 10.1093/nar/27.4.1063
ISSN: 1362-4962
Divisions: Current > QUT Faculties and Divisions > Science & Engineering Faculty
Deposited On: 22 Apr 2014 04:51
Last Modified: 21 Jun 2017 00:02

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