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Nucleosome periodicity in HeLa cell chromatin as probed by micrococcal nuclease
Abstract:
When HeLa cell nuclei were treated with micrococcal nuclease (nucleate 3-oligonucleotidohydrolase, EC 3.1.4.7), lysed, and centrifuged, the supernatant from early digests contained two predominant classes of polynucleosomes of repeat size 8N and 16N. With increasing digestion time, the 16 N polynucleosome appeared to be cleaved to the 8N species and finally to the basic subunit of chromatin. The size of the polynucleosomes has been determined by DNA analysis and on polyacrylamide electrophoretic gels of native chromatin particles. The 16N polynucleosome appears to be a unique higher ordered structural component of HeLa cell chromatin. Our recent report, showing that the nuclear protein-modifying enzyme poly(ADP-ribose) polymerase increases in specific activity progressively with increasing nucleosome repeat size up to 8-10N, has been extended in the present study. Activity was also elevated in the polynucleosomes of the 16N structure preferentially cleaved by micrococcal nuclease, although specific activity of the enzyme was highest in octanucleosomes. Acceptors for poly(ADP-ribose) have also been determined in these particles.
Insights
HeLa cell chromatin contains unique 16N polynucleosomes that are cleaved into smaller units. Poly(ADP-ribose) polymerase activity is highest in octanucleosomes within these structures.
Area of Science:
- Molecular Biology
- Chromatin Structure
- Biochemistry
Background:
- Chromatin is organized into nucleosomes, the basic repeating units of DNA and histone proteins.
- Higher-order chromatin structures influence gene regulation and DNA accessibility.
- Poly(ADP-ribose) polymerase (PARP) is a nuclear enzyme involved in DNA repair and other cellular processes.
Purpose of the Study:
- To investigate the structural organization of HeLa cell chromatin using micrococcal nuclease digestion.
- To characterize the size and properties of polynucleosomes.
- To examine the relationship between polynucleosome structure and poly(ADP-ribose) polymerase activity.
Main Methods:
- HeLa cell nuclei were treated with micrococcal nuclease.
- Nuclei were lysed and centrifuged to isolate polynucleosomes.
- Polynucleosome sizes were determined by DNA analysis and gel electrophoresis.
- Poly(ADP-ribose) polymerase activity and acceptors were analyzed in isolated chromatin particles.
Main Results:
- Micrococcal nuclease digestion yielded predominant 8N and 16N polynucleosome classes.
- The 16N polynucleosome was observed to be a unique higher-order structure, progressively cleaved to 8N and then to the basic chromatin subunit.
- Poly(ADP-ribose) polymerase specific activity increased with polynucleosome size up to 8-10N, with elevated activity in 16N structures, and highest activity in octanucleosomes.
Conclusions:
- HeLa cell chromatin exhibits a distinct higher-order structure represented by the 16N polynucleosome.
- The 16N polynucleosome is susceptible to micrococcal nuclease digestion, yielding smaller units.
- Poly(ADP-ribose) polymerase activity is modulated by chromatin structure, with preferential association with larger polynucleosome assemblies.