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Related Experiment Videos

Structural characterization of separated H DNA conformers.

J N Glover1, C S Farah, D E Pulleyblank

  • 1Department of Biochemistry, University of Toronto, Ontario, Canada.

Biochemistry
|December 18, 1990
PubMed
Summary

Polypyrimidine/polypurine DNA sequences form two distinct protonated triplex structures (H DNA) under stress. These structures differ in stability and helical unwinding, with one form showing greater stability due to specific base pairing interactions.

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Area of Science:

  • Molecular Biology
  • Biophysics
  • Structural Biology

Background:

  • Polypyrimidine/polypurine DNA sequences can form unusual DNA structures like H DNA.
  • These structures arise under specific conditions, including negative superhelical stress and low pH.
  • A d(TC)17-d(GA)17 insert serves as a model system to study these phenomena.

Purpose of the Study:

  • To investigate the structural and energetic properties of two isomeric protonated triplex DNA forms.
  • To understand the relationship between helical unwinding, stability, and DNA sequence.
  • To elucidate the molecular basis for the differential stability of these H DNA conformers.

Main Methods:

  • Separation of topoisomeric variants using agarose gel electrophoresis.
  • Assessment of DNA structure and accessibility using chemical probes (permanganate and acid-induced depurination).

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  • Development of topological models to analyze helical unwinding and twist.

    • Main Results:

    • Two distinct H DNA isomers (H-y5 and H-y3) were identified, differing in helical unwinding.
    • The H-y5 isomer, where the 5'-half of the d(GA)n strand participates in the triplex, is more stable at low superhelix densities.
    • Structural analysis revealed differences in solvent accessibility and helical twist between the isomers, with H-y5 being less accessible.

    Conclusions:

    • The stability of H DNA isomers is dependent on the degree of helical unwinding and superhelix density.
    • The H-y5 isomer's enhanced stability is attributed to Watson-Crick base pairing within the pyrimidine loop interacting with the exiting d(GA)n strand.
    • These findings provide insights into the structural plasticity of DNA and the formation of non-canonical structures under physiological stress.