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Electron microscopic identification of supercoiled regions in complex DNA structures
Journal of Molecular Biology
|January 20, 1987
Summary
Psoralen-UV treatment creates DNA crosslinks, revealing structural differences. Supercoiled DNA remains intact upon heating, while discontinuous DNA denatures, indicating distinct structural states.
Area of Science:
- Molecular Biology
- Biophysics
- Genetics
Background:
- DNA structure and topology are crucial for various cellular processes.
- Psoralen-UV treatment is a method for crosslinking DNA strands.
- Supercoiled DNA exhibits unique physical and chemical properties compared to relaxed DNA.
Purpose of the Study:
- To investigate the impact of psoralen-UV crosslinking on DNA structure, specifically differentiating between supercoiled and covalently discontinuous regions.
- To explore how DNA superhelicity influences psoralen intercalation and subsequent crosslinking.
- To establish methods for distinguishing supercoiled from discontinuous DNA domains.
Main Methods:
- Treatment of lambda DNA replicative intermediates (theta structures) and plasmid circles with psoralen and long-wavelength UV light.
- Purification, denaturation, and electron microscopy of treated DNA structures.
- Thermal denaturation experiments on crosslinked and linearized DNA molecules.
- Analysis of crosslinking density in relation to DNA superhelicity.
Main Results:
- Psoralen-UV treatment of theta structures resulted in native parental DNA segments and denatured daughter segments after thermal denaturation, suggesting differential structural integrity.
- Supercoiled plasmid circles remained native after psoralen-UV treatment and denaturation due to enhanced thermal stability.
- Linearized DNA molecules, treated post-linearization, showed higher crosslinking density, indicating superhelicity influences psoralen intercalation.
- Two distinct properties were identified: differential thermal denaturation and increased crosslinking density in supercoiled regions.
Conclusions:
- Supercoiled DNA domains retain their native structure upon thermal treatment after psoralen-UV crosslinking, while covalently discontinuous regions denature.
- Negative superhelicity enhances psoralen intercalation, leading to a higher crosslinking density in these regions.
- Crosslinking density serves as a molecular memory of the superhelical state, even after supercoiling is lost.