DNA-loop formation on nucleosomes shown by in situ scanning force microscopy of supercoiled DNA.
The flexibility of the chromatin structure, necessary for the processing of the genomic DNA, is controlled by a number of factors where flexibility and mobility of the nucleosomes is essential. Here, the influence of DNA supercoiling on the structure of single nucleosomes is investigated. Circular supercoiled plasmid DNA sub-saturated with histones was visualized by scanning force microscopy (SFM) in aqueous solution. SFM-imaging compared with topological analysis indicates instability of nucleosomes when the salt concentration is raised from 10 mM to 100 mM NaCl. Nucleosomes were observed after the deposition to the used scanning surface, i.e. mica coated with polylysine. On the images, the nucleosomes appear with a high probability in end-loops near the apices of the superhelices. In 100 mM NaCl but not in 10 mM NaCl, a significant number of complexes present the nucleosomes on superhelical crossings mainly located adjacent to an end-loop. The morphology of these structures and statistical analysis suggest that DNA loops were formed on the histone octamers, where the loop size distribution shows a pronounced peak at 50 nm. Recently, the formation and diffusion of loops on octamers has been discussed as a mechanism of translocations of nucleosomes along DNA. The presented data likely confirm the occurrence of loops, which may be stabilized by supercoiling. Analysis of the structure of regular nucleosomes not located on crossings indicates that reducing the salt concentration leads to more conformations, where DNA is partially unwrapped from the distal ends of the octamer.
Bussiek
Malte
M
German Cancer Research Center, Div. Biophysics of Macromolecules, Im Neuenheimer Feld 580, D-69120 Heidelberg, Germany. m.bussiek@dkfz.de
Tóth
Katalin
K
Brun
Nathalie
N
Langowski
Jörg
J
D004278:DNA, Superhelical / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D006657:Histones / Q000378:metabolism; D018625:Microscopy, Atomic Force; D009690:Nucleic Acid Conformation; D009707:Nucleosomes / Q000737:chemistry* / Q000378:metabolism / Q000648:ultrastructure*; D010957:Plasmids / Q000737:chemistry / Q000378:metabolism / Q000648:ultrastructure
D004278:DNA, Superhelical; D006657:Histones; D009707:Nucleosomes