Interaction of enhancer-binding protein EBP1 (NF-kappa B) with the human immunodeficiency virus type 1 enhancer.
Human EBP1, isolated from HeLa cells, binds to a 10-base-pair (bp) sequence in cellular and viral enhancers that is also recognized by the inducible transcription factor NF-kappa B. Here we describe the interaction of purified EBP1 with the 10-bp repeated sequence that is responsive to signals which activate T cells and which form part of the human immunodeficiency virus type 1 (HIV-1) enhancer. DNase I footprinting indicates that both 10-bp sites on the same molecule, located between -80 and -105 on the HIV-1 long terminal repeat, can be occupied by EBP1, while dimethyl sulfate protection and methylation interference experiments indicate which purine bases are in contact with the protein. The presence of bases which exhibit increased rates of dimethyl sulfate-induced methylation in the presence of EBP1 indicate that interaction of EBP1 with its recognition site is accompanied by distortion of the DNA double helix. Supporting this conclusion is the observation that the polyamine spermidine dramatically increases EBP1 binding to its cognate site on the DNA. Studies with human T cells (Jurkat) and nucleotide stimulation data suggest that EBP1 is the activated form of NF-kappa B in these cells.
Department of Biochemistry and Microbiology, University of St. Andrews, Scotland, United Kingdom.
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Clark
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Matthews
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Hay
D001483:Base Sequence; D004268:DNA-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D004742:Enhancer Elements, Genetic; D005814:Genes, Viral; D015497:HIV-1 / Q000235:genetics* / Q000378:metabolism; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D016328:NF-kappa B; D015345:Oligonucleotide Probes / Q000138:chemical synthesis; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D014157:Transcription Factors / Q000302:isolation & purification / Q000378:metabolism*
D004268:DNA-Binding Proteins; D016328:NF-kappa B; D015345:Oligonucleotide Probes; D014157:Transcription Factors