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Function of the N‐terminal segment of the RecA‐dependent nuclease Ref
Author(s) -
Dvorak Rachel,
Gruber Angela,
Olsen Tayla,
Cox Michael
Publication year - 2015
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.29.1_supplement.879.7
Subject(s) - nuclease , cleavage (geology) , endonuclease , dna , chemistry , recombination , truncation (statistics) , amino acid , bacteriophage , microbiology and biotechnology , stereochemistry , biology , biochemistry , escherichia coli , mathematics , gene , paleontology , statistics , fracture (geology)
The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA‐dependent, HNH endonuclease. It can be directed to create targeted double‐strand breaks within a displacement loop formed by RecA. The 76 amino acid N‐terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N‐terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization, and is necessary for efficient Ref‐mediated DNA cleavage. Specifically, Ref N‐terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA‐mediated targeting nucleases. We propose a Ref cleavage model, in which the N‐terminal region acts as an anchor and allows rearrangement of the nuclease domain for sequential cleavage of strands in double‐stranded DNA.