Nudeosomes Are the Building Blocks of Chromosomes
The majority of the DNA in eukaryotic cells is packaged into nucleo-somes. The nucleosome is composed of a core of eight his tunc proteins and the DNA wrapped around them. The DNA between each nucleo-some (the "siring" in the "beads on a string") is called linker DNA, By assembling into nudeosomes, the DNA is compacted approximately sixfold. This is far short of the 1,000- to 10,000-fold DNA compaction observed in eukaryotic cells. Nevertheless, this first stage of DNA packaging is essential for all the remaining levels of DNA compaction.
The DNA most tightly associated with the nucleosome, called the core DNA, is wound approximately 1.B5 limes around the uutside of the histune ociamer like thread around a spool [Figure 7-18), The length of DNA associated with each nucleosome can be determined using nuclease treatment (Box 7-1, Micrococcal Nuclease and the DNA Associated with the Nucleosome). The -147 base pair length of this DNA is an invariant feature of nucleosomes in all eukaryotic cells. In contrast, the length of the linker DNA between iiucleosomes is variable. Typically this distance is 20-60 bp and each eukaryote has a characteristic average linker DNA length (Table 7-4). The difference in average linker DNA length is likely to reflect the differences in the nature of larger structures formed by nucleosotnal DNA
FIGURE 7-18 DNA packaged into nudeosomes. (a) Schematic of the packag mg and organization of nudeosomes. (b) Crystal structure of a nucleosome shewing DNA wrapped around the btstone protein core. H2A is si own in red, H2B in yeltuw, H3 in purple, end H4 in green, Note that the colors of the different histone proteins here and in following structures are the same. (Luger K., Miider AW, Richmond R.K., Sargent D.F, and Richmond TJ. 1997. Nature 389: 25) -260.) image prepared with BobScript, MolScript, and Raster 3D.
nucleosome histone core core DNA (146 bp)
nucleosome histone core core DNA (146 bp)
Box 7-1 Micrococcal Nuclease and the DNA Associated with the Nucteosome
Nudeosomes were first purified by treating chromosomes with a sequence nonspecific nuclease called micrococcal nuclease. The ability of this enzyme to cleave DMA is primarily governed by the accessibility of the DNA. Thus, micrococcal nudease cleaves protein-free DNA sequences rapidly and protein-associated DNA sequences poorly. Limited treatment of chromosome?; with this enzyme results in a nuclease-resistant population of DNA molecules that are associated with h ¡stones. These DNA molecules are between 160-220 base pairs in length and are associated with two copies each of histones H2A, H2B. H3, and H4. On average, these particles indude the DNA tightly associated with the nucteosome as well as one unit of linker DNA. More extensive micrococcal nuclease treatment degrades all of the linker DNA The remaining minimal nudeosome includes only 147 bp of DNA and is called the nucteosome core particle.
The average length nf DNA associated with each nucteosome can be measured in a simple experiment (Box 7-1 Figure 1). Chromatin is treated with the enzyme microaxxal nuclease but this time ( inly gently. This results in single cuts in some but not all of the linker DNA After nuclease treatment, the DNA is extracted from all proteins (including the histones) and subjected to gel electrophoresis to separate the DNA by size. Flectropborests reveals a "ladder" of fragments that are multiples of the average nudecsoine-to-nudeasome distance. A ladder of fragments is observed because the micrococcal nudease-treated chromatin fe only partially digested. Thus, sometimes multiple nudeosomes wdl remain unseparated by digestion, leadfng to DNA fragments equivalent to all the DNA bound by these nucleosomes. Further digestion would result in at) linker DNA being cleaved and the formation of nucleosome core partides and a single -147 bp fragment.
400 bp more extensive digestion
200 bp more extensive digestion
gel electrophoresis bp 800 600
-11 nm released nucteosome core particle released nucteosome core particle bp 800 600
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