title: Combined micrococcal nuclease and exonuclease III digestion reveals precise positions of the nucleosome core/linker junctions: implications for high-resolution nucleosome mapping.
abstract: Micrococcal nuclease (MNase) is extensively used in genome-wide mapping of nucleosomes but its preference for AT-rich DNA leads to errors in establishing precise positions of nucleosomes. Here, we show that the MNase digestion of nucleosomes assembled on a strong nucleosome positioning sequence, Widom's clone 601, releases nucleosome cores whose sizes are strongly affected by the linker DNA sequence. Our experiments produced nucleosomal DNA sizes varying between 147 and 155 bp, with positions of the MNase cuts reflecting positions of the A⋅T pairs rather than the nucleosome core/linker junctions determined by X-ray crystallography. Extent of chromatosomal DNA protection by linker histone H1 also depends on the linker DNA sequence. Remarkably, we found that a combined treatment with MNase and exonuclease III (exoIII) overcomes MNase sequence preference producing nucleosomal DNA trimmed symmetrically and precisely at the core/linker junctions regardless of the underlying DNA sequence. We propose that combined MNase/exoIII digestion can be applied to in situ chromatin for unbiased genome-wide mapping of nucleosome positions that is not influenced by DNA sequences at the core/linker junctions. The same approach can be also used for the precise mapping of the extent of linker DNA protection by H1 and other protein factors associated with nucleosome linkers.
authors: Laboratory of Cell Biology, CCR, National Cancer Institute, NIH, Bethesda, MD, USA.
authors: Tatiana
authors: T
authors: Nikitina
authors: Laboratory of Cell Biology, CCR, National Cancer Institute, NIH, Bethesda, MD, USA.
authors: Difei
authors: D
authors: Wang
authors: Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA.
authors: Misha
authors: M
authors: Gomberg
authors: Department of Biochemistry and Molecular Biology, Pennsylvania State University College of Medicine, Hershey, PA, USA. Electronic address: sag17@psu.edu.
authors: Sergei A
authors: SA
authors: Grigoryev
authors: Laboratory of Cell Biology, CCR, National Cancer Institute, NIH, Bethesda, MD, USA. Electronic address: zhurkin@nih.gov.
authors: Victor B
authors: VB
authors: Zhurkin
mesh_terms: D001483:Base Sequence; D004247:DNA / Q000737:chemistry*; D005090:Exodeoxyribonucleases / Q000378:metabolism*; D008836:Micrococcal Nuclease / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009707:Nucleosomes / Q000737:chemistry*
chemical_list: D009707:Nucleosomes; D004247:DNA; D005090:Exodeoxyribonucleases; C020133:exodeoxyribonuclease III; D008836:Micrococcal Nuclease