Distortion of the DNA double helix by RAP1 at silencers and multiple telomeric binding sites.
Repressor Activator Protein 1 (RAP1) is an essential nuclear protein of the yeast Saccharomyces cerevisiae that recognizes a 13 base-pair (bp) consensus sequence found in numerous upstream activating sequences, at the silencers of transcriptionally repressed mating-type genes, and in telomeric tracts, called (C1-3 A) repeats. RAP1 has been shown to influence transcriptional activation, transcriptional repression, telomere length, circular plasmid segregation and meiotic recombination in vivo. We have studied the structure of the protein-DNA complex reconstituted in vitro with highly purified RAP1, by using DNase I and chemical footprinting. Both full-length RAP1 and its minimal DNA-binding domain of roughly 30 kDa, induce a distortion within the 13 bp recognition site, as demonstrated by reactivity to KMnO4 primarily at nucleotides 8 and 10 in the binding consensus Rc/AAYCCRYNCAYY. Dimethylsulphate reactivity shows that RAP1 binding does not create unpaired regions at its binding site, although the DNA may be locally underwound or aberrantly base-paired at the permanganate reactive nucleotides. In addition to the permanganate-sensitive distortion, the full-length RAP1, but not its DNA-binding domain, induces a bend in DNA 5' of the recognition sequence, altering the electrophoretic mobility of the protein-DNA complex. The KMnO4-reactivity has allowed a precise mapping of RAP1 molecules on telomeric DNA, revealing RAP1 sites as frequently as one per 18 bp of telomeric DNA, or potentially 20 RAP1 molecules bound per average telomeric tract of 370 bp. This suggests that RAP1 plays a major role in organizing yeast telomeres, and is consistent with recently published immunofluorescence studies showing a major fraction of RAP1 at the ends of meiotic chromosomes.
Swiss Institute for Experimental Cancer Research (ISREC), Epalinges's Lausanne.
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Gilson
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Roberge
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Giraldo
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Rhodes
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Gasser
D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D004271:DNA, Fungal / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D004121:Dimethyl Sulfoxide / Q000494:pharmacology; D006834:Hydrazines / Q000494:pharmacology; D008969:Molecular Sequence Data; D009690:Nucleic Acid Conformation; D011196:Potassium Permanganate / Q000494:pharmacology; D012045:Regulatory Sequences, Nucleic Acid; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D016615:Telomere / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism*
D004271:DNA, Fungal; D004268:DNA-Binding Proteins; D006834:Hydrazines; D014157:Transcription Factors; D011196:Potassium Permanganate; C029424:hydrazine; D004121:Dimethyl Sulfoxide