Trajectory of nucleosomal linker DNA studied by fluorescence resonance energy transfer

While the structure of the nucleosome core is known in atomic detail, the p recise geometry of the DNA beyond the core particle is still unknown. We ha ve used fluorescence resonance energy transfer (FRET) for determining the e nd-to-end distance of DNA fragments assembled with histones into nucleosome s. The DNA of a length of 150-220 bp was labeled with rhodamine-X on one en d and fluorescein or Alexa 488 on the other. Assembling nucleosomes on thes e DNA fragments leads to a measurable energy transfer. The end-to-end dista nce computed from the FRET increases from 60 +/- 5 Angstrom at 150 bp to 75 +/- 5 Angstrom at 170 bp without measurable change above it. These distanc es are compatible with different geometries of the linker DNA, all having i n common that no crossing can be observed up to 220 bp. Addition of H1 hist one leads to an increase in energy transfer, indicating a compaction of the linker DNA toward the nucleosome.