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Ultrastructural studies on chromatin digestion by micrococcal nuclease in the presence of polyamines
Abstract:
Nuclei purified from C57BL mouse submandibular salivary gland were treated with a range of micrococcal nuclease concentrations and times of treatment (from 0.5 unit for 2.5 min to 50 units for 30 min) in the presence of polyamines. About 50% of the chromatin was solubilized initially but with prolonged digestion this chromatin became insoluble again. Electron microscopy showed destruction of the finely dispersed chromatin with mild digestion, followed by aggregation of chromatin with more vigorous digestion. The early disappearance of finely dispersed chromatin filaments was not accompanied by preferential solubilization of chromatin associated with RNA polymerase II (euchromatin). These data suggest that the polyamines markedly reduce the susceptibility of euchromatin to micrococcal nuclease digestion.
Insights
Polyamines protect mouse euchromatin from micrococcal nuclease digestion. This suggests polyamines reduce chromatin susceptibility to enzymatic breakdown, impacting DNA accessibility.
Area of Science:
- Molecular Biology
- Chromatin Structure
- Biochemistry
Background:
- Chromatin structure regulates DNA accessibility and gene expression.
- Micrococcal nuclease (MNase) is used to probe chromatin accessibility.
- Polyamines are cations that interact with DNA and chromatin.
Purpose of the Study:
- To investigate the effect of polyamines on micrococcal nuclease digestion of chromatin.
- To determine if polyamines alter the susceptibility of euchromatin to MNase.
Main Methods:
- Nuclei were isolated from C57BL mouse submandibular salivary glands.
- Nuclei were treated with varying concentrations and times of micrococcal nuclease in the presence of polyamines.
- Chromatin solubility was assessed, and structural changes were observed using electron microscopy.
Main Results:
- Initial MNase digestion solubilized approximately 50% of chromatin.
- Prolonged digestion led to chromatin insolubility.
- Electron microscopy revealed chromatin aggregation with increased digestion.
- Euchromatin, associated with RNA polymerase II, was not preferentially solubilized.
- Polyamines significantly decreased euchromatin susceptibility to MNase digestion.
Conclusions:
- Polyamines markedly reduce the susceptibility of euchromatin to micrococcal nuclease digestion.
- This suggests polyamines play a role in modulating chromatin structure and DNA accessibility.