Protein binding to simple repetitive sequences depends on DNA secondary structure(s).
Simple repetitive DNA sequences are ubiquitous constituents of eukaryotic chromosomes. The properties of simple repeats generate increased interest as expansions of certain trinucleotide blocks cause human diseases. We studied protein binding and structural features of (gaa x ttc)n tracts e.g. in the polymorphic frataxin intron 1 and (gt)n(ga)m stretches from different HLA-DRB1 alleles in their original genomic environments. Electrophoretic mobility shift assays revealed that HeLa nuclear proteins bind to DNA fragments containing these simple repeat blocks. The major retarded protein/DNA complexes comprise, in both cases, zinc-dependent proteins present in nuclear extracts from different cell types. Competition experiments using various simple repeats differing in length and flanking regions demonstrate specific interactions. DNase I footprinting shows protein-binding sites located either within the repeats alone or within the repeats as well as their flanking regions, often with preference for one strand. Comparing different (gt)n(ga)m alleles, a regular pattern of footprints was not detectable in the (gt)n part indicating that the zinc-dependent protein recognizes structural rather than sequence-specific features. OsO4 and DEPC modifications followed by electrophoretic and electron microscopical analyses demonstrate that the homopurine blocks often form different types of intramolecular triple helices. A similar situation was evident using (gaa x ttc)n blocks of different lengths within frataxin intron 1 as targets. These data have functional implications for non-coding (gaa x ttc)n and (gt)n(ga)m tracts with regard to gene expression in vivo.
Molecular Human Genetics, Ruhr-University, Bochum, Germany.
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Mäueler
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Bassili
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Epplen
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HG
Keyl
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JT
Epplen
D000818:Animals; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D015394:Molecular Structure; D009690:Nucleic Acid Conformation; D011485:Protein Binding; D012091:Repetitive Sequences, Nucleic Acid; D015032:Zinc / Q000737:chemistry
D004268:DNA-Binding Proteins; D004247:DNA; D015032:Zinc