title: Function of the N-terminal segment of the RecA-dependent nuclease Ref.
abstract: The bacteriophage P1 Ref (recombination enhancement function) protein is a RecA-dependent, HNH endonuclease. It can be directed to create targeted double-strand breaks within a displacement loop formed by RecA. The 76 amino acid N-terminal region of Ref is positively charged (25/76 amino acid residues) and inherently unstructured in solution. Our investigation of N-terminal truncation variants shows this region is required for DNA binding, contains a Cys involved in incidental dimerization and is necessary for efficient Ref-mediated DNA cleavage. Specifically, Ref N-terminal truncation variants lacking between 21 and 47 amino acids are more effective RecA-mediated targeting nucleases. We propose a more refined set of options for the Ref-mediated cleavage mechanism, featuring the N-terminal region as an anchor for at least one of the DNA strand cleavage events.
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Angela J
authors.initials: AJ
authors.lastname: Gruber
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Tayla M
authors.initials: TM
authors.lastname: Olsen
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA.
authors.forename: Rachel H
authors.initials: RH
authors.lastname: Dvorak
authors.affiliation: Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, USA cox@biochem.wisc.edu.
authors.forename: Michael M
authors.initials: MM
authors.lastname: Cox
mesh_terms: D000595:Amino Acid Sequence; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D005454:Fluorescence Polarization; D008969:Molecular Sequence Data; D011938:Rec A Recombinases / Q000737:chemistry / Q000378:metabolism*; D017386:Sequence Homology, Amino Acid
chemical_list: D004220:Disulfides; D011938:Rec A Recombinases