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Defining the cut site specificity of Ref – a RecA dependent endonuclease
Author(s) -
Wan Yui Chun Serena,
Ronayne Erin A,
Cox Michael M
Publication year - 2016
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.30.1_supplement.794.1
Subject(s) - nuclease , cleavage (geology) , endonuclease , dna , protein subunit , nucleotide , cleave , chemistry , biophysics , biology , biochemistry , gene , paleontology , fracture (geology)
Recombination enhancement function (Ref) is a RecA‐dependent endonuclease that creates double strand breaks within in a displacement loop of the dsDNA substrate. DNA cleavage is not random, and we are currently deciphering the precise cleavage specificity of the Ref nuclease. Cleavage occurs throughout the D‐loop, although some locations are clearly preferred. The overall cleavage patterns of Ref are highly repeatable, indicating that the process is likely influenced by sequence and/or protein interactions. When reactions are carried out with ATPase deficient RecA or with non‐hydrolyzable ATP analog so that RecA filaments are static, patterns with preferred cleavage peaks recurring every three nucleotides are observed. As the binding site of a RecA subunit is three nucleotides or base pairs, these results suggest that cleavage may be directed by, or occur at, the subunit‐subunit interface of a RecA filament. This DNA cleaving system has the targeting potential of the CRISPR system in genome editing, although the complexity of the Ref system currently limits its application. This project is aimed at a further understanding of the nuclease mechanism of Ref, protein‐protein interactions between Ref and RecA, and will be useful for future applications of the Ref‐RecA system.