Interaction of nucleosomes with oligonucleotides and minor groove binding ligands
Interaction of nucleosomes with oligonucleotides and minor groove binding ligands
We have used DNase 1 footprinting to examine the formation of triplexes on DNA fragments which have been complexed with nucleosome core particles. For these studies we used the 160 base pair tyrT DNA sequence, mutating this to introduce 12-14 base oligopurine tracts as different positions. By targeting these different sites with triplex forming oligonucleotides we have shown that the location of the target site on the nucleosome-bound DNA is critical for determining triplex formation. If the target site is located within the central 80 base pairs of the nucleosomal DNA then triplex formation is inhibited. However triplex formation is permitted as the target site is moved towards the end of the nucleosomal DNA. Longer triplexes (up to 25 bases) show a similar behaviour.
Similar experiments used DNA sequences containing long poly(dA).poly(dT) tracts, examining their interaction with oligo(dT). In this case we are able to demonstrate stable triplex formation on nucleosome-bound DNA fragments, most likely because of the unusual structure of poly(dA).poly(dT) tracts.
A separate study investigated the interaction of PNA (peptide nucleic acid) with free and nucleosome-bound DNA. We show the formation of both 2:1 and 1:2 PNA:DNA complexes, involving strand invasion and triplex formation respectively, on free DNA. In contrast no interaction was detected with nucleosome bound DNA.
The effects of a AT-selective minor groove binding ligands on nucleosome-bound DNA fragments has also been investigated by footprinting. The AT-rich binding sites for these ligands are preferentially oriented so as to face towards the nucleosome surface and so should be protected from drug binding. Hydroxyl radical and DNase 1 cleavage patterns of reconstituted DNA fragments suggest that these ligands cause DNA to rotate by 180o on the nucleosome surface.
University of Southampton
1997
Brown, Philip Michael
(1997)
Interaction of nucleosomes with oligonucleotides and minor groove binding ligands.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
We have used DNase 1 footprinting to examine the formation of triplexes on DNA fragments which have been complexed with nucleosome core particles. For these studies we used the 160 base pair tyrT DNA sequence, mutating this to introduce 12-14 base oligopurine tracts as different positions. By targeting these different sites with triplex forming oligonucleotides we have shown that the location of the target site on the nucleosome-bound DNA is critical for determining triplex formation. If the target site is located within the central 80 base pairs of the nucleosomal DNA then triplex formation is inhibited. However triplex formation is permitted as the target site is moved towards the end of the nucleosomal DNA. Longer triplexes (up to 25 bases) show a similar behaviour.
Similar experiments used DNA sequences containing long poly(dA).poly(dT) tracts, examining their interaction with oligo(dT). In this case we are able to demonstrate stable triplex formation on nucleosome-bound DNA fragments, most likely because of the unusual structure of poly(dA).poly(dT) tracts.
A separate study investigated the interaction of PNA (peptide nucleic acid) with free and nucleosome-bound DNA. We show the formation of both 2:1 and 1:2 PNA:DNA complexes, involving strand invasion and triplex formation respectively, on free DNA. In contrast no interaction was detected with nucleosome bound DNA.
The effects of a AT-selective minor groove binding ligands on nucleosome-bound DNA fragments has also been investigated by footprinting. The AT-rich binding sites for these ligands are preferentially oriented so as to face towards the nucleosome surface and so should be protected from drug binding. Hydroxyl radical and DNase 1 cleavage patterns of reconstituted DNA fragments suggest that these ligands cause DNA to rotate by 180o on the nucleosome surface.
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Published date: 1997
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Local EPrints ID: 462982
URI: http://eprints.soton.ac.uk/id/eprint/462982
PURE UUID: 75c5953e-8f96-4fce-8a30-65dbe4fefaf4
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Date deposited: 04 Jul 2022 20:34
Last modified: 04 Jul 2022 20:34
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Author:
Philip Michael Brown
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