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.
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.
Wang
Jin K
JK
Department of Biomedical Engineering, Chien-Shiung Wu Laboratory, Southeast University, Nanjing 210096, China.
Li
Tong X
TX
Bai
Yun F
YF
Lu
Zu H
ZH
D001665:Binding Sites; D003461:Crystallography; D004268:DNA-Binding Proteins / Q000737:chemistry*; D019281:Dimerization; D024202:Electrophoretic Mobility Shift Assay; D020672:I-kappa B Proteins / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D016328:NF-kappa B / Q000032:analysis / Q000737:chemistry* / Q000235:genetics; D052002:NF-kappa B p50 Subunit; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods
D004268:DNA-Binding Proteins; D020672:I-kappa B Proteins; D016328:NF-kappa B; D052002:NF-kappa B p50 Subunit