Opening of the nucleosome structure is essential for accessing genomic DNA. To study the mechanism of this process, we monitor the distance between various fluorescently labeled positions on mononucleosomes by single-molecule Förster resonance energy transfer (FRET). Here, we compare nucleosomes reconstituted from recombinant mouse, Xenopus, and yeast histones. As DNA sequences we compared, the effect of 5S rDNA, MMTV-B sequence, and Widom 601 DNA. The stability, as measured by the salt concentration at the opening transition midpoint, is lowest for yeast, followed by Xenopus and mouse. The 601 DNA sequence builds much more stable nucleosomes and the distribution of FRET efficiencies is narrower than for those reconstituted on 5S rDNA or MMTV-B sequences. The opening pathway through an intermediate state, as found for Xenopus histones, could be verified for the mouse and yeast systems and for the different DNA sequences, suggesting a general mechanism for accessing nucleosomal DNA.
Keywords: chromatin structure; resonance energy transfer; salt destabilization; single molecule spectroscopy.
Copyright © 2013 International Society for Advancement of Cytometry.