Creating targeted double‐stranded breaks in vitro and in vivo using RecA‐dependent endonucleases P1 and φW39 Ref (552.1)

Abstract

P1 bacteriophage recombination enhancement function (Ref) protein is an HNH class and RecA‐dependent endonuclease. RecA‐bound oligonucleotides homologous to a specific region on circular dsDNA will catalyze RecA strand‐invasion, creating a displacement loop (D‐loop). Upon Ref addition, double‐stranded breaks (DSBs) are created within the D‐loop. Thus, the Ref protein, in concert with RecA, is a programmable endonuclease. The closely related φW39 Ref protein exhibits increased RecA‐dependent endonuclease activity compared to P1 Ref. P1 Ref protein missing the disordered 76 N‐terminus residues is incapable of DNA binding and exhibits decreased nuclease activity, which indicates the importance of the N‐terminal region for these functions. Characterization of N‐terminal truncations based on charge distribution for φW39 Refs ΔN21, ΔN47, ΔN59, ΔN66, and ΔN74 revealed modulated activity for DSB formation and DNA binding. In addition to these in vitro studies, cells transformed with a plasmid containing P1 Ref and introduced to an oligo homologous to a region in the rpsL gene demonstrate recombination events with the oligo. The Ref/RecA/oligo system acts as an oligo‐targeting inducer of DSBs operating both in vitro and in vivo with potential for genome editing functions and in vitro targeting.Grant Funding Source: NIH GM32335