Trajectory of nucleosomal linker DNA studied by fluorescence resonance energy transfer.
While the structure of the nucleosome core is known in atomic detail, the precise geometry of the DNA beyond the core particle is still unknown. We have used fluorescence resonance energy transfer (FRET) for determining the end-to-end distance of DNA fragments assembled with histones into nucleosomes. The DNA of a length of 150-220 bp was labeled with rhodamine-X on one end and fluorescein or Alexa 488 on the other. Assembling nucleosomes on these DNA fragments leads to a measurable energy transfer. The end-to-end distance computed from the FRET increases from 60 +/- 5 A at 150 bp to 75 +/- 5 A at 170 bp without measurable change above it. These distances are compatible with different geometries of the linker DNA, all having in common that no crossing can be observed up to 220 bp. Addition of H1 histone leads to an increase in energy transfer, indicating a compaction of the linker DNA toward the nucleosome.
Tóth
K
K
Division of Biophysics of Macromolecules (H0500), Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany. kati.toth@dkfz.de
Brun
N
N
Langowski
J
J
D000818:Animals; D019556:COS Cells; D004247:DNA / Q000737:chemistry* / Q000378:metabolism; D053938:DNA Fragmentation; D004735:Energy Transfer; D019793:Fluorescein / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006657:Histones / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D006834:Hydrazines / Q000737:chemistry; D008956:Models, Chemical; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism; D013050:Spectrometry, Fluorescence / Q000379:methods; D014981:Xenopus
C417561:Alexa 488 hydrazide; D005456:Fluorescent Dyes; D006657:Histones; D006834:Hydrazines; D009707:Nucleosomes; D004247:DNA; D019793:Fluorescein