Evaluating the binding affinities of NF-kappaB p50 homodimer to the wild-type and single-nucleotide mutant Ig-kappaB sites by the unimolecular dsDNA microarray

Anal Biochem. 2003 May 15;316(2):192-201. doi: 10.1016/s0003-2697(03)00049-6.

Abstract

This study investigated the binding affinities of NF-kappaB p50 homodimer to the wild-type and single-nucleotide mutant Ig-kappaB sites by the unimolecular dsDNA microarray which was fabricated with a novel scheme. The importance of each nucleotide of Ig-kappaB site for the sequence-specific p50p50/Ig-kappaB interaction was thus evaluated. The results demonstrate that the nucleotides at different positions contribute differently to the p50p50/Ig-kappaB binding interaction. The G(1), G(2), and C(10) are most important for p50p50/Ig-kappaB binding interaction and determine the specificity of p50p50/Ig-kappaB interaction, which replacements with any other nucleotide could result in the similarly greatest binding affinity losses. Comparatively, the G(3), A(4), T(8), and C(9) are less important for p50p50/Ig-kappaB interaction and regulate the binding affinity, which substitutions with the variant nucleotide could change the binding affinity differently. The C(5) is least important for p50p50/Ig-kappaB interaction, the randomized nucleotide exchange of which little affects on p50p50/Ig-kappaB binding affinity. Among all possible single-nucleotide mutants, the T(8) to C mutation could strengthen p50p50/Ig-kappaB interaction. The T(7) acts differently from its symmetric C(5) and the axial T(6) is necessary for high-affinity p50p50/Ig-kappaB interaction. The unimolecular dsDNA microarray provides a reliable method for exploring the binding affinities of DNA-binding proteins with a larger number of DNA targets.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Binding Sites
  • Crystallography
  • DNA-Binding Proteins / chemistry*
  • Dimerization
  • Electrophoretic Mobility Shift Assay
  • I-kappa B Proteins / analysis
  • I-kappa B Proteins / chemistry*
  • I-kappa B Proteins / genetics
  • Mutation
  • NF-kappa B / analysis
  • NF-kappa B / chemistry*
  • NF-kappa B / genetics
  • NF-kappa B p50 Subunit
  • Oligonucleotide Array Sequence Analysis / methods

Substances

  • DNA-Binding Proteins
  • I-kappa B Proteins
  • NF-kappa B
  • NF-kappa B p50 Subunit