35 research outputs found
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On the location of the linker histones and the linker DNA in the 30 nm fiber of chromatin
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
Single-Molecule Approaches Reveal the Idiosyncrasies of RNA Polymerases
Recently developed single-molecule techniques have provided new insights into the function of one of the most complex and highly regulated processes in the cell—the transcription of the DNA template into RNA. This review discusses methods and results from this emerging field, and it puts them in perspective of existing biochemical and structural data
Steric exclusion and wrapping of the excluded DNA strand occurs along discrete external binding paths during MCM helicase unwinding
The minichromosome maintenance (MCM) helicase complex is essential for the initiation and elongation of DNA replication in both the eukaryotic and archaeal domains. The archaeal homohexameric MCM helicase from Sulfolobus solfataricus serves as a model for understanding mechanisms of DNA unwinding. In this report, the displaced 5′-tail is shown to provide stability to the MCM complex on DNA and contribute to unwinding. Mutations in a positively charged patch on the exterior surface of the MCM hexamer destabilize this interaction, alter the path of the displaced 5′-tail DNA and reduce unwinding. DNA footprinting and single-molecule fluorescence experiments support a previously unrecognized wrapping of the 5′-tail. This mode of hexameric helicase DNA unwinding is termed the steric exclusion and wrapping (SEW) model, where the 3′-tail is encircled by the helicase while the displaced 5′-tail wraps around defined paths on the exterior of the helicase. The novel wrapping mechanism stabilizes the MCM complex in a positive unwinding mode, protects the displaced single-stranded DNA tail and prevents reannealing