Understanding the structure of the 30 nm fiber in chromatin is relevant to understanding eukaryotic replication and transcription. The major controversy among the models of the fiber concerns the disposition of the linker DNA, the DNA between adjacent nucleosomes, and the location of the linker histones. To determine if the location of the linker histones and the linker DNA was internal or external, chromatin was digested with immobilized proteases and nucleases. The chromatin was probed either in a low salt extended 10 nm fiber of nucleosomes or in progressive compactions (addition of increasing amounts of salt) to form a condensed (30 nm) fiber.
Digestion experiments performed on linker histones either in chicken erythrocyte chromatin, or free in solution or bound in mononucleosomes revealed: (1) Histone H5 is more protected than histone H1 in the fiber; (2) The N-and C-terminal portions of H1 do not change their
accessibility upon compaction of the fiber; the tails of H5, however, become significantly internalized in the 30 nm fiber; (3) phenylalanine in the globular domain of both H1 and H5 is inaccessible both in the fiber and in mononucleosomes. Sedimentation velocity measurements demonstrate that the conformation of the fiber at all its different condensation states is highly sensitive to cuts in even a few of the linker histone molecules.
The structure of these chromatin fibers has also been probed using micrococcal nuclease, both membrane-immobilized and free in solution, under extremely mild digestion conditions. The linker DNA is almost completely protected against digestion in the 30 nm fibers, whereas it is readily accessible in the more extended structures, independent of whether immobilized or free enzyme is employed. To circumvent complications due to the sensitivity of the enzyme to the salt concentration, control experiments were performed in which chromatin fibers were glutaraldehyde-fixed under different ionic conditions and then digested in low salt. The results were very similar to the above. Experiments with fibers of intermediate degree of condensation revealed a direct relationship between the degree of compaction and the resistance of linker DNA to digestion. These results support models for chromatin structure in which access to the linkers is limited by local steric hindrance, rather than by internalization in the core of the fibers.
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