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Nucleosome "phasing" and cruciform structures in circular supercoiled pBR322 DNA.
Cell Biophysics
|March 1, 1984
Summary
Cruciform DNA structures were found in supercoiled plasmid DNA, even when associated with histone octamers. These structures, located near AT-rich regions, may protrude from nucleosomes, explaining their stability.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Supercoiled DNA can adopt non-B DNA structures, such as cruciforms.
- Nucleosomes can influence DNA conformation and stability.
- The pBR322 plasmid is a common tool in molecular biology research.
Purpose of the Study:
- To investigate the presence and location of cruciform structures in pBR322 supercoiled DNA.
- To determine if histone octamers affect cruciform formation.
- To propose a model for cruciform stability in the context of nucleosomes.
Main Methods:
- S1 endonuclease cleavage to detect cruciform structures.
- EcoRI restriction enzyme analysis.
- DNA sequence analysis to identify AT-rich regions.
- Restriction enzyme analysis for nucleosome phasing.
Main Results:
- Cruciform structures were detected in naked and histone-complexed pBR322 supercoiled DNA.
- S1-hypersensitive sites, indicative of cruciforms, were located near AT-rich regions.
- Nucleosome phasing was observed in these AT-rich regions.
- A model was proposed where cruciforms protrude from the nucleosome surface.
Conclusions:
- Cruciform structures can form in pBR322 supercoiled DNA.
- Nucleosome formation does not preclude cruciform stability and may even stabilize them.
- Cruciform structures protruding from nucleosomes explain their existence under conditions of relaxed superhelicity.