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DNA knotting abolishes in vitro chromatin assembly
1Departamento de Biología Molecular y Celular, Centro de Investigación y Desarrollo, CSIC, Jordi Girona, 18 E-08034, Barcelona, Spain.
The Journal of Biological Chemistry
|June 14, 1996
Summary
Yeast topoisomerase II in vitro can create DNA topological knots. These knots are untied by the enzyme, and knotted DNA prevents nucleosome assembly, suggesting a role in chromatin regulation.
Area of Science:
- Molecular Biology
- Biochemistry
- Genetics
Background:
- Topological knots in DNA can be formed and manipulated by enzymes.
- Topoisomerase II is crucial for managing DNA topology.
- Nucleosome assembly is essential for DNA packaging and regulation.
Purpose of the Study:
- To investigate the in vitro formation and resolution of DNA topological knots using yeast topoisomerase II.
- To determine the effect of DNA knotting on nucleosome assembly.
- To elucidate the role of topoisomerase II in chromatin assembly regulation.
Main Methods:
- In vitro DNA knotting assay using purified yeast topoisomerase II and double-stranded DNA.
- Agarose gel electrophoresis for topological analysis of DNA knots.
- Nucleosome assembly assay using Xenopus oocyte S-150 extract and topological DNA templates.
- Micrococcal nuclease analysis to assess chromatin structure.
Main Results:
- Yeast topoisomerase II efficiently produces DNA topological knots in an ATP-dependent manner.
- Topoisomerase II, but not topoisomerase I, can resolve these DNA knots.
- Knotted DNA templates inhibit in vitro nucleosome assembly, while relaxed DNA does not.
- Pretreatment of knotted DNA with topoisomerase II restores canonical minichromosome assembly.
Conclusions:
- Topoisomerase II plays a significant role in the dynamic formation and resolution of DNA topological knots.
- DNA knotting by topoisomerase II can regulate chromatin assembly.
- A mechanism for topoisomerase II-directed chromatin assembly regulation is proposed.