Nucleic Acids Research, Vol 26, Issue 11 2526-2535, Copyright © 1998 by Oxford University Press
DJ Fitzgerald and JN Anderson
A computational study was previously carried out to analyze DNA sequences
that are known to position histone octamers at single translational sites.
A conserved pattern of intrinsic DNA curvature was uncovered that was
proposed to direct the formation of nucleosomes to unique positions. The
pattern consists of two regions of curved DNA separated by preferred
lengths of non-curved DNA. In the present study, 11 synthetic DNAs were
constructed which contain two regions of curved DNA of the form
[(A5.T5)(G/C)5]4 separated by non-curved regions of variable length.
Translational mapping experiments of in vitro reconstituted mononucleosomes
using exonuclease III, micrococcal nuclease and restriction enzymes
demonstrated that two of the fragments positioned nucleosomes at a single
site while the remaining fragments positioned octamers at multiple sites
spaced at 10 base intervals. The synthetic molecules that positioned
nucleosomes at a single site contain non-curved central regions of the same
lengths that were seen in natural nucleosome positioning sequences.
Hydroxyl radical and DNase I digests of the synthetic DNAs in reconstituted
nucleosomes showed that the synthetic curved element on one side of the
nucleosomal dyad assumed a rotational orientation where narrow minor
grooves of the A- tracts faced the histone surface with all molecules. In
contrast, the curved element on the other side of the nucleosome displayed
variable rotational orientations between molecules which appeared to be
related to the positioning effect. These results suggest that asymmetry
between the two halves of nucleosomal DNA may facilitate translational
positioning.
ARTICLES
Unique translational positioning of nucleosomes on synthetic DNAs
Department of Biological Sciences, Purdue University, West Lafayette, IN 47907-1392, USA.
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