Recently, a highly repetitive DNA sequence family (GRS) from tobacco was described in our laboratory. These sequences were found to be localized predominantly in the pericentromeric heterochromatin of tobacco chromosomes. To test the hypothesis that these sequences play an important role in the formation of heterochromatin, we investigated the DNA curvature of the GRS sequences and its possible impact to the chromatin structure at these loci. Application of the nearest-neighbour wedge model of intrinsic DNA curvature for the GRS1 family member predicted two loci of curvature: a major bend at the 5' end of the sequence and a minor bend of opposite direction at the centre of the GRS1. The presence of the major and the minor loci of DNA curvature was studied experimentally using permutation analysis and site-directed mutagenesis. The experimental results were consistent with the computer predictions. We gave evidence that the described DNA curvature is also present in the entire GRS family. Genomic statistical sequencing showed the conservation of the major bend sequence determinants in the members of the GRS family. To investigate the chromatin structure at the GRS sequences, we determined the nucleosome positioning in vivo at these sequences using thermal cycle primer extension. A relation between the curvature pattern and the histone octamer position was observed: the major bend is excluded from the nucleosome surface to the linker region, while the minor bend is distributed along the core DNA. The suggestion is made that the sequences in the minor locus of curvature define the rotational setting of the nucleosome, and a possible role of the major bend as a factor, which defines the translational setting, is discussed.

Institute of Biophysics Academy of Sciences of the Czech Republic Brno

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