DNA structure in nucleosome positioning
Abstract
In the present work, satellite DNA sequences were shown to display wide variation in magnitude of intrinsic bending. However, a conserved pattern of bending was seen in all 57 satellite sequences that were studied. The pattern consisted of repeating units of two 50–60 base pair bending elements which were separated by a 20–30 base-pair region of low curvature. Sequences which have been shown to position nucleosomes at single major sites were also examined and a pattern of DNA curvature resembling the two peak units seen in satellites was observed. In order to test the involvement of the conserved curvature pattern in nucleosome positioning, eleven synthetic DNAs were constructed which each contain two regions of curved DNA of the form ((A5-T 5)(G/C)5)4 separated by non-curved regions of variable length. Translational mapping experiments of in vitro reconstituted mononucleosomes demonstrated that two of the synthetic fragments positioned nucleosomes at a single site while the remaining fragments positioned octamers at multiple sites spaced at 10 base-pair intervals. In support of the curvature-based positioning model, the synthetic molecules that positioned nucleosomes at a single site were those that contained non-curved central regions of the same lengths that were seen in natural nucleosome positioning sequences. Sequence-dependent DNA distortion was advanced as a possible explanation for the positioning effect. Therefore, chemical probes were utilized to identify sites of DNA distortion in the synthetic positioning sequences. A permanganate hypersite was identified in the positioning sequences 15 base-pairs from the nucleosorne pseudo-dyad at a site known to display DNA distortion in the mature nucleosome. A second generation of synthetic molecules were constructed which contained substitutions in the hypersite region and tested for affinity for histone octamers and translational positioning in in vitro studies. The results revealed that as little as a single base substitution in the hypersite region dramatically affected affinity and positioning activity. A reconstituted 5S rDNA positioning sequence from L. variegatus was shown to display a permanganate hypersite 16 base-pairs from its pseudo-dyad which suggests that DNA distortion might be involved in nucleosome positioning on natural DNAs. An examination of sequence properties of natural positioning sequences strengthens this proposition.
Degree
Ph.D.
Advisors
Anderson, Purdue University.
Subject Area
Mechanical engineering|Molecules|Mechanics
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