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Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system

Maher, L. James, III and Dervan, Peter B. and Wold, Barbara (1992) Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system. Biochemistry, 31 (1). pp. 70-81. ISSN 0006-2960. doi:10.1021/bi00116a012.

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A site-specific triple-helical DNA complex has previously been shown to inhibit DNA binding by eukaryotic transcription factor Sp1. To examine the functional consequences of such inhibition, homopurine target sequences for oligonucleotide-directed triple-helix formation were inserted in various configurations relative to Sp1 transcription activator binding sites, upstream of the TATA element of recombinant eukaryotic promoters. The resulting promoters were tested for activity in the presence or absence of recombinant human Sp1 in a Drosophila in vitro transcription system lacking endogenous Sp1. When triple-helical complexes were assembled on the promoters by incubation with specific oligodeoxyribonucleotides, promoter-specific repression of basal transcription was observed in the absence of Sp1. Transcriptional repression required the preassembly of triple-helical complexes before addition of nuclear extract. The degree of basal repression was a function of the number and proximity of triple-helical complexes relative to the basal promoter complex. Repression did not result from triple-helix-induced template degradation. Addition of recombinant Sp1 did not cause derepression. These results suggest that triple-helical complexes can repress transcription primarily by blocking promoter DNA assembly into initiation complexes rather than by occluding Sp1 binding. One of several plausible mechanisms for triple-helix-induced repression involves changes in DNA flexibility. Evidence in favor of this model is provided by a permutation-dependent gel mobility assay in which formation of site-specific triple-helical complexes is shown to stiffen double-helical DNA.

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Dervan, Peter B.0000-0001-8852-7306
Wold, Barbara0000-0003-3235-8130
Additional Information:© 1992 American Chemical Society. Received June 19, 1991; Revised Manuscript Received September 4, 1991. Supported by research grants from the National Institutes of Health to P.B.D. (GM-35724) and to B.W. (AR-40780) and the Beckman Institute of the California Institute of Technology to B.W. and an American Cancer Society Postdoctoral Fellowship to L.J.M.
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Caltech Beckman InstituteUNSPECIFIED
American Cancer SocietyUNSPECIFIED
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Record Number:CaltechAUTHORS:20160510-145808159
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Official Citation:Analysis of promoter-specific repression by triple-helical DNA complexes in a eukaryotic cell-free transcription system L. James Maher III, Peter B. Dervan, and Barbara Wold Biochemistry 1992 31 (1), 70-81 DOI: 10.1021/bi00116a012
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:66951
Deposited By: Victoria Brennan
Deposited On:18 May 2016 23:44
Last Modified:11 Nov 2021 00:24

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