Specificity of Mammalian Y-box Binding Protein p50 in Interaction with ss and ds DNA Analyzed with Generic Oligonucleotide Microchip

p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4096 sin...

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Bibliographic Details
Published in:Journal of molecular biology 2002-11, Vol.324 (1), p.73-87
Main Authors: Zasedateleva, O.A, Krylov, A.S, Prokopenko, D.V, Skabkin, M.A, Ovchinnikov, L.P, Kolchinsky, A, Mirzabekov, A.D
Format: Article
Language:English
Subjects:
Animals
biochip
DNA
DNA - metabolism
DNA, Single-Stranded - metabolism
Electrophoretic Mobility Shift Assay
Image Processing, Computer-Assisted
interaction
Mammals
Oligonucleotide Array Sequence Analysis - methods
Oligonucleotides - chemistry
Oligonucleotides - metabolism
p50
Repressor Proteins - genetics
Repressor Proteins - metabolism
RNA-Binding Proteins - genetics
RNA-Binding Proteins - metabolism
Substrate Specificity
Temperature
Y-box
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Summary:p50 protein is a member of the Y-box binding transcription factor family and is a counterpart of YB-1 protein. The generic microchip was used to analyze the sequence specificity of p50 binding to single (ss) and double-stranded (ds) oligodeoxyribonucleotides. The generic microchip contained 4096 single-stranded octadeoxyribonucleotides in which all possible core 6-mers (4 6=4096) were flanked at their 3′ and 5′-ends with degenerated nucleotides. The oligonucleotides were chemically immobilized within polyacrylamide gel pads fixed on a glass slide. The binding of p50 to the generic microchip was shown to be the most specific to ss GGGG motif and then to ss CACC and CATC motifs. GC-rich ds oligonucleotides of the generic microchip, and particularly those containing GGTG/CACC, GATG/CATC, and GTGG/CCAC heterogeneous motifs, were most efficiently destabilized due to interaction with p50. Gel-shift electrophoresis has shown that the protein exhibits much higher binding specificity to 24-mer oligoA-TGGGGG-oligoA containing G-rich 6-mer, in comparison with 24-mer oligoA-AAATAT-oligoA carrying A,T-rich 6-mer in full correspondence with the data obtained with the microchip. Studies of DNA-binding proteins using gel-immobilized ss and ds DNA fragments provide a unique possibility to detect low-affinity complexes of these proteins with short sequence motifs and assess the role of these motifs in sequence-specific interactions with long recognition sites.
ISSN:0022-2836
1089-8638
DOI:10.1016/S0022-2836(02)00937-3