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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
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.
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+).
- 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.