NoRC binds to the entry/exit sites of the nucleosome.
(A) Overview of the experimental approach. (B) Analytical EMSA of the DNase I footprinting reaction. For further analysis of the DNase I digestion pattern the nucleosome and nucleosome/NoRC complexes were isolated from the gel. The arrow indicates the NoRC/nucleosome complexes. (C) DNase I footprinting of DNA and centrally positioned nucleosomes. A 247 bp rDNA promoter fragment (−231 to +16 respective to the start site) was radioactively labelled either at the 5′ or 3′ end. The free DNA (bar) and the centrally positioned nucleosome (gray ellipse) were treated with DNase I and after 10 sec and 30 sec the reactions were stopped with EDTA. Nucleosomes and DNA were resolved by EMSA and the bands were isolated. Purified DNA was subsequently analysed on 7% sequencing gels. A scheme of the central positioned nucleosome is shown on the right. (D) Recombinant NoRC was incubated with a purified nucleosome positioned at the center of the DNA fragment and partially digested with DNase I (10 and 30 sec). The reaction was stopped by the addition of EDTA and the nucleoprotein complexes were separated by native gel electrophoresis. Nucleosomes and NoRC/nucleosome complexes were isolated, DNA purified and analysed on 7% sequencing gels. The nucleosome position (gray ellipse) and the radioactive end-labeling (32P) are indicated. Changes in the digestion pattern upon NoRC treatment are marked with a gray bar, significant changes are highlighted with stars.
