Nuclear factor binding to a DNA sequence element that represses MMTV transcription induces a structural transition and leads to the contact of single-stranded binding proteins with DNA.
NRE1 is a DNA sequence element in the long terminal repeat of mouse mammary tumor virus through which viral transcription is repressed. In addition to double-stranded DNA binding, both upper- and lower-stranded NRE1 binding activities occur in nuclear extracts. All three binding activities appear to be important for transcriptional effects. We report that occupancy of NRE1 within linear double-stranded NRE1 induces a structural transition in upstream flanking DNA that is facilitated by Mg2+. This transition was reflected by the striking DNase I sensitivity of the DNA. As Mg2+ concentration was increased, discrete DNase I hypersensitivity on one face of the DNA progressed to complete degradation of template. On the DNA face opposite the DNase I hypersensitivity, Mg2+ promoted regularly spaced cleavage by the single-strand-specific cleavage agents KMnO4 and S1 nuclease. Induction of degradation by DNase I occurred independently of MMTV sequences flanking NRE1, because nuclear extract-dependent DNase I sensitivity was conferred to an unrelated DNA fragment by introduction of a 23-bp NRE1-containing oligonucleotide. UV protein-DNA cross-linking revealed that addition of Mg2+ to a double-stranded NRE1 DNA binding assay induced conversion from a double- to a single-stranded protein-DNA cross-linking pattern. Thus, nuclear factor binding to NRE1 induces changes in DNA topology that promote the direct contact of single-stranded NRE1 binding factors with DNA.
Department of Medicine, University of Ottawa, Loeb Institute for Medical Research, Ottawa Civic Hospital, Ontario, Canada.
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W
Giffin
R J
RJ
Haché
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004247:DNA / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003850:Deoxyribonuclease I / Q000378:metabolism; D006801:Humans; D006868:Hydrolysis; D008274:Magnesium / Q000378:metabolism; D008324:Mammary Tumor Virus, Mouse / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D012091:Repetitive Sequences, Nucleic Acid; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D004247:DNA; D003850:Deoxyribonuclease I; D008274:Magnesium