Abstract
Simple repeating d(GA.TC)n DNA sequences are frequently found at eukaryotic promoters, and in some cases they have been shown to be nucleosome free in vivo. These sequences show a high degree of structural polymorphism and are capable of adopting several types of non-B-DNA conformations. Here we show that the structural versatility of these sequences affects their ability to be packed into nucleosomes. Nucleosome assembly onto short double-stranded DNA fragments carrying d(GA.TC)n sequences of different length (n = 10 and n = 22) is very efficient. However, when the simple repeating sequence is forming a [CT(GA.TC)] triplex, nucleosome assembly is either prevented, as in the case of the d(GA.TC)22 sequence, or results in the destabilization of the triple-stranded conformation, as in the case of the d(GA.TC)10 sequence. Similarly, formation of triple-stranded DNA is hindered when the sequence is organized as nucleosomes. Efficient formation of triplex DNA occurs only at relatively high ionic strength (0.6 M NaCl), when the nucleosome is partially destabilized, and results in the disruption of the nucleosomal particle. These results indicate that nucleosome assembly and triplex formation are competing processes.
Highlights
From the structural point of view, d(GA1⁄7TC)n DNA sequences show a remarkable high degree of polymorphism
Nucleosome Assembly at d(GA1⁄7TC)n Sequences Is Inhibited by the Formation of Stable [CT(GA1⁄7TC)]n Intermolecular Triplexes—Mononucleosomes were reconstituted in vitro onto short DNA fragments carrying d(GA1⁄7TC)n sequences of different length (n ϭ 10 and n ϭ 22) (Fig. 1)
The rotational setting is better for the GA10 fragment than for the GA22 fragment, suggesting that the repeated sequence does not determine the setting
Summary
DNAs and Triplex Formation—The different d(GA1⁄7TC)n DNA sequences studied in this paper were inserted in a pUC18 plasmid carrying a modified polylinker. DNA fragments containing the sequences of interest (Fig. 1) were excised from the plasmid by digestion with EcoRV and XhoI and were purified by electrophoresis on 1% agarose gels. These fragments were 3Ј end labeled at the XhoI site. When the effect of nucleosome structure on the formation of triple-stranded DNA was analyzed, the same molar excess of CT-oligonucleotide was incubated with the reconstituted DNA fragment in a buffer containing 10 mM Tris-HCl (pH 7), 1 mM EDTA, 0.01 mM phenylmethylsulfonyl fluoride, 0.1 mM dithiothreitol, and the amount of NaCl indicated in each case.
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