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Electron microscopic imaging of supercoiled DNA containing highly biased sequence
M Kato1, T D Gurkov, G E Elmesiry
1Department of Life Sciences, C.I.A.S., Osaka Prefecture University, Sakai 599-8531, Japan.
Nucleic Acids Research. Supplement (2001)
|July 3, 2003
Summary
Electron microscopy revealed unusual DNA structures in pUC19 derivatives. GC-rich sequences and pyrimidine/purine-biased DNA segments formed distinct triplex-like stem structures under superhelical tension.
Area of Science:
- Molecular Biology
- Biophysics
- Genetics
Background:
- Superhelical tension in DNA can induce non-canonical structures.
- Previous S1 nuclease assays indicated unusual DNA conformations in specific pUC19 derivatives.
- Understanding DNA structure is crucial for gene regulation and stability.
Purpose of the Study:
- To visualize and characterize unusual DNA structures formed by pUC19 derivatives with specific sequence motifs.
- To correlate DNA sequence characteristics with observed structural conformations.
- To investigate the role of GC-rich and pyrimidine/purine-biased sequences in DNA structure formation.
Main Methods:
- Electron microscopic imaging of DNA molecules.
- Preparation of pUC19 derivatives with defined sequences (pTIR10, pTIS303, pBan1).
- Analysis of DNA unfolding, twisting, and entanglement under specific grid-spreading conditions.
Main Results:
- Electron microscopy visualized unfolded, twisted, and entangled DNA molecules.
- Approximately 70% of unfolded pTIR10 molecules exhibited triplex-like stems (18-38 nm).
- pTIS303 displayed slightly shorter stem structures compared to pTIR10, suggesting sequence-dependent structure formation.
Conclusions:
- The study confirms the formation of unusual DNA structures, including triplex-like stems, in pUC19 derivatives.
- GC-rich segments appear to be key determinants for forming these unusual DNA structures.
- DNA sequence composition significantly influences conformational adaptability under superhelical stress.