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Related Experiment Videos

Characterization of a bimobile DNA junction.

M Lu1, Q Guo, J E Mueller

  • 1Department of Chemistry, New York University, New York 10003.

The Journal of Biological Chemistry
|October 5, 1990
PubMed
Summary

Chemical and enzymatic footprinting revealed dynamic structural changes in branched DNA junctions. The branch site showed altered reactivity and cleavage patterns, indicating flexibility and cation-dependent structural transitions.

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Area of Science:

  • Molecular Biology
  • Biochemistry
  • Structural Biology

Background:

  • Branched DNA structures are crucial intermediates in genetic recombination and replication.
  • Understanding the dynamic nature of DNA junctions is key to elucidating these processes.
  • Previous studies focused on immobile junctions, leaving dynamic aspects less explored.

Purpose of the Study:

  • To investigate the chemical and enzymatic reactivity of a mobile branched DNA junction.
  • To characterize the structural dynamics and cation-dependent alterations of the DNA junction.
  • To compare the behavior of a mobile junction with previously studied immobile analogs.

Main Methods:

  • Chemical footprinting using MPE.Fe(II) and (OP)2Cu(I) reagents.
  • Enzymatic footprinting with resolvase enzymes (endonuclease VII from phage T4 and endonuclease I from phage T7).

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  • Analysis of DNA junction structure in the presence and absence of divalent cations (Mg2+).

    • Main Results:

    • Fe(II).EDTA showed protection of residues at the branch point.
    • Resoluase enzymes cleaved all four strands near the branch.
    • MPE.Fe(II) and (OP)2Cu(I) revealed hyper-reactivity at the branch site, involving more residues than in immobile junctions.
    • Absence of divalent cations led to structural alterations, loss of enhanced cleavage, and increased reactivity of purines to diethyl pyrocarbonate.
    • In the presence of Mg2+, three migrational isomers coexisted, likely forming 2-fold symmetric structures.

    Conclusions:

    • The mobile branched DNA junction exhibits dynamic structural properties distinct from immobile junctions.
    • Divalent cations like Mg2+ stabilize specific conformations of the branched DNA structure.
    • The findings provide insights into the structural flexibility and reactivity of DNA junctions during biological processes.