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Related Experiment Videos

Hairpin opening by single-strand-specific nucleases

E B Kabotyanski1, C Zhu, D A Kallick

  • 1Department of Microbiology and Immunology, Baylor College of Medicine, Houston, TX 77030, USA.

Nucleic Acids Research
|October 11, 1995
PubMed
Summary

DNA hairpin ends, crucial intermediates in V(D)J recombination, are opened by specific nucleases. The loop sequence of these hairpins dictates where enzymes like P1 nuclease and mung bean nuclease cleave, revealing structural insights.

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Area of Science:

  • Molecular Biology
  • Genetics
  • Biochemistry

Background:

  • DNA hairpin ends are likely intermediates in V(D)J recombination, a key process in immune system development.
  • Current models suggest hairpin opening generates single-stranded extensions that form P nucleotides at recombination junctions.
  • The enzymatic activities responsible for hairpin opening remain largely uncharacterized.

Purpose of the Study:

  • To investigate the enzymatic activities responsible for opening DNA hairpin ends.
  • To explore how the sequence within the hairpin loop influences the hairpin opening reaction.
  • To identify structural features of hairpin loops recognized by single-strand-specific nucleases.

Main Methods:

  • Utilized two single-strand-specific nucleases: P1 nuclease and mung bean nuclease (MBN).

Related Experiment Videos

  • Analyzed the cleavage patterns of 22 different DNA hairpin sequences.
  • Correlated nuclease digestion patterns with existing Nuclear Magnetic Resonance (NMR) structural data for selected hairpin loops.

    • Main Results:

    • Both P1 nuclease and mung bean nuclease effectively opened various hairpin ends, leaving short (1-2 nucleotide) single-stranded extensions.
    • The terminal 4 nucleotides of the hairpin loop sequence were found to significantly influence the sites of enzymatic cleavage.
    • Structural data (NMR) provided insights into how these nucleases recognize specific structural features within the hairpin loops.

    Conclusions:

    • Single-strand-specific nucleases can open DNA hairpins, generating the short single-stranded extensions implicated in P nucleotide formation.
    • The sequence and resulting structure of the hairpin loop are critical determinants of cleavage site specificity.
    • This study offers new understanding of the structural basis for nuclease recognition and activity at DNA hairpin ends.