skip to main content
OSTI.GOV title logo U.S. Department of Energy
Office of Scientific and Technical Information

Title: Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-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 {angstrom} 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 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 introductionmore » of directed, recombinogenic double-strand breaks.« less

Authors:
; ; ; ; ; ;  [1]
  1. UW
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
OTHERNIH
OSTI Identifier:
1034567
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.
Additional Journal Information:
Journal Volume: 286; Journal Issue: (10) ; 03, 2011; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AMINO ACIDS; BACTERIOPHAGES; CLEAVAGE; DNA; DNA REPAIR; ENDONUCLEASES; HISTIDINE; IN VIVO; NUCLEASES; NUCLEIC ACIDS; NUCLEOPROTEINS; PROTEINS; RECOMBINATION; RESIDUES; RESOLUTION; SUBSTRATES

Citation Formats

Gruenig, Marielle C, Lu, Duo, Won, Sang Joon, Dulberger, Charles L, Manlick, Angela J, Keck, James L, and Cox, Michael M. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1. United States: N. p., 2012. Web. doi:10.1074/jbc.M110.205088.
Gruenig, Marielle C, Lu, Duo, Won, Sang Joon, Dulberger, Charles L, Manlick, Angela J, Keck, James L, & Cox, Michael M. Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1. United States. https://doi.org/10.1074/jbc.M110.205088
Gruenig, Marielle C, Lu, Duo, Won, Sang Joon, Dulberger, Charles L, Manlick, Angela J, Keck, James L, and Cox, Michael M. Fri . "Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1". United States. https://doi.org/10.1074/jbc.M110.205088.
@article{osti_1034567,
title = {Creating Directed Double-strand Breaks with the Ref Protein: A Novel Rec A-Dependent Nuclease from Bacteriophage P1},
author = {Gruenig, Marielle C and Lu, Duo and Won, Sang Joon and Dulberger, Charles L and Manlick, Angela J and Keck, James L and Cox, Michael M},
abstractNote = {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 {angstrom} 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 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.},
doi = {10.1074/jbc.M110.205088},
url = {https://www.osti.gov/biblio/1034567}, journal = {J. Biol. Chem.},
issn = {0021-9258},
number = (10) ; 03, 2011,
volume = 286,
place = {United States},
year = {2012},
month = {3}
}