title: Creating directed double-strand breaks with the Ref protein: a novel RecA-dependent nuclease from bacteriophage P1.
abstract: 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; that is, 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 Å resolution. The core structure, consisting of residues 77-186, consists of a central 2-stranded β-hairpin that is sandwiched between several α-helical and extended loop elements. The N-terminal 76 amino acid residues are disordered; this flexible region is required for optimal activity. The overall structure of Ref, including several putative active site histidine residues, defines a new subclass of HNH-family nucleases. We propose that enhancement of recombination by Ref reflects the introduction of directed, recombinogenic double-strand breaks.
authors.affiliation: From the Department of Biochemistry, University of Wisconsin and.
authors.forename: Marielle C
authors.initials: MC
authors.lastname: Gruenig
authors.affiliation: the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706.
authors.forename: Duo
authors.initials: D
authors.lastname: Lu
authors.affiliation: From the Department of Biochemistry, University of Wisconsin and.
authors.forename: Sang Joon
authors.initials: SJ
authors.lastname: Won
authors.affiliation: From the Department of Biochemistry, University of Wisconsin and.
authors.forename: Charles L
authors.initials: CL
authors.lastname: Dulberger
authors.affiliation: From the Department of Biochemistry, University of Wisconsin and.
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Manlick
authors.affiliation: the Department of Biomolecular Chemistry, University of Wisconsin School of Medicine and Public Health, Madison, Wisconsin 53706.
authors.forename: James L
authors.initials: JL
authors.lastname: Keck
authors.affiliation: From the Department of Biochemistry, University of Wisconsin and. Electronic address: cox@biochem.wisc.edu.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Cox
mesh_terms: D017101:Bacteriophage P1 / Q000201:enzymology* / Q000235:genetics; D053903:DNA Breaks, Double-Stranded; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011938:Rec A Recombinases / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship; D014764:Viral Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D029968:Escherichia coli Proteins; D014764:Viral Proteins; D011938:Rec A Recombinases; D003851:Deoxyribonucleases