DNA sequence and structural requirements in nucleosome positioning
Abstract
A previous report demonstrated that one site in a nucleosome assembled onto a synthetic positioning sequence known as Fragment 67 is hypersensitive to permanganate. The site is required for positioning activity and is located 1.5 turns from the dyad, which is a region of high DNA curvature in the nucleosome. Here, the permanganate sensitivity of the nucleosome positioning Fragment 601 was examined in order to expand the dataset of nucleosome sequences containing KMnO4 hypersensitive sites. The hyperreactive T residue in the six sites detected as well as the one in Fragment 67 and three in the 5 S rDNA positioning sequence were contained within a TA step. Seven of the ten sequences were of the form CTAGPuG or the related sequence TTAAPu. These motifs were also found in the binding sites of several transcriptional regulatory proteins that kink DNA. In order to assess the significance of these sites, the 10 bp positioning determinant in Fragment 67 was removed and replaced by the nine sequences from the 5S rDNA and Fragment 601. These derivative fragments resulted in high nucleosome stability and positioning as compared to a control sequence that contained an AT step in place of the TA step. Single base-pair substitutions at the TA-step in Fragment 67 revealed that stability and positioning activity followed the order: TA > TG > TT ≥ TC ≈ GG ≈ GA ≈ AT. Sequences flanking the TA step were also shown to be critical for nucleosome stability and positioning. Nucleosome positioning was restored to near wild-type levels with (CTG)3, which can form slipped stranded structures and with one base bulges that kink DNA. The results of this study suggest that nucleosome positioning determinants are at least 4 bp long in contrast to existing models which are based on dinucleotide parameters. The results show that single base-pair changes at these positioning determinants could have profound effects on those genomic functions that depend on ordered nucleosomes.
Degree
Ph.D.
Advisors
Anderson, Purdue University.
Subject Area
Molecular biology|Biochemistry
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