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Related Experiment Videos

DNA structure specificity of Rap endonuclease.

G J Sharples1, L M Corbett, P McGlynn

  • 1Institute of Genetics, University of Nottingham, Queens Medical Centre, Nottingham NG7 2UH, UK. gary.sharples@nottingham.ac.uk

Nucleic Acids Research
|October 16, 1999
PubMed
Summary

Phage lambda's Rap endonuclease specifically nicks branched DNA, favoring Holliday junctions and D-loops crucial for phage recombination. This enzyme requires metal ions like manganese for efficient DNA cleavage.

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Area of Science:

  • Molecular Biology
  • Structural Biology
  • Biochemistry

Background:

  • Phage lambda's Rap protein is an endonuclease known to interact with branched DNA structures.
  • Rap's role in nicking D-loops during phage recombination has been proposed, potentially bypassing Holliday junctions.

Purpose of the Study:

  • To investigate the structure specificity of the Rap endonuclease.
  • To determine the preferred DNA substrates and cleavage sites of Rap.
  • To understand Rap's role in phage recombination intermediates.

Main Methods:

  • Synthesis of various branched DNA molecules by annealing partially complementary oligonucleotides.
  • Assays to determine Rap endonuclease activity on different DNA structures in the presence of Mg(2+) or Mn(2+).

Related Experiment Videos

  • Mapping of endonuclease incision sites on various branched DNA substrates.

    • Main Results:

    • Rap endonuclease requires divalent metal ions (Mg(2+) or Mn(2+)), with Mn(2+) enhancing cleavage 5-fold.
    • Rap preferentially cleaves at the branch point of D-loop, bubble, flayed duplex, 5'-flap, and Y junction DNA substrates.
    • Rap exhibits high structure specificity, preferring 4- and 3-stranded DNA (resembling D-loops and Holliday junctions) over other structures.

    Conclusions:

    • Rap endonuclease displays a strong preference for DNA structures characteristic of recombination intermediates, such as D-loops and Holliday junctions.
    • The enzyme's specificity suggests a key role in processing these branched DNA structures during phage lambda recombination.
    • Rap's ability to nick branched DNA at specific sites highlights its importance in resolving complex DNA structures.