The RecA-dependent endonuclease Ref: characterization of DNA binding, nuclease activity, and use for in vivo genome editing in Escherichia coli

Abstract

Bacteriophage Ref protein is an HNH class, RecA-dependent endonuclease. RecA-bound oligonucleotides complementary to a region on circular dsDNA will catalyze RecA strand-invasion, creating a displacement loop (D-loop). Ref can be directed to create double-strand breaks within the D-loop based only on the sequence of the oligonucleotide used. The Ref protein consists of a globular domain containing the active-site and 76 disordered N-tenninal residues responsible for DNA binding. Characterization ofNterminal truncations revealed modulated activity for DNA binding and double-strand break formation, this system has been explored in vivo. Escherichia coli containing RecA, Ref, and a targeting oligonucleotide demonstrate oligo-recombination induced by DSBs created by Ref; this has potential for being implemented in genome editing.