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Publication Abstract from PubMed

The bacteriophage P1-encoded Ref protein enhances RecA-dependent recombination in vivo by an unknown mechanism. We demonstrate that Ref is a new type of enzyme - a RecA-dependent nuclease. Ref binds to ss and dsDNA, but does not cleave any DNA substrate until RecA protein and ATP are added to form RecA nucleoprotein filaments. Ref cleaves only where RecA protein is bound. RecA functions as a co-nuclease in the Ref/RecA system. Ref nuclease activity can be limited to the targeted strands of short RecA-containing D-loops. The result is a uniquely programmable endonuclease activity, producing targeted double strand breaks at any chosen DNA sequence in an oligonucleotide-directed fashion. We present evidence indicating that cleavage occurs in the RecA filament groove. The structure of the Ref protein has been determined to 1.4 angstroms resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded beta-hairpin that is sandwiched between several alpha-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for activity. The overall structure of Ref, including several putative active site Histidine residues, defines a new sub-class of HNH-family nucleases. We propose that enhancement of recombination by Ref in bacteria reflects the introduction of directed, recombinogenic double strand breaks.

Creating directed double strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.,Gruenig MC, Lu D, Won SJ, Dulberger CL, Manlick AJ, Keck JL, Cox MM J Biol Chem. 2011 Jan 3. PMID:21193392^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.