title: The monomeric GIY-YIG homing endonuclease I-BmoI uses a molecular anchor and a flexible tether to sequentially nick DNA.
abstract: The GIY-YIG nuclease domain is found within protein scaffolds that participate in diverse cellular pathways and contains a single active site that hydrolyzes DNA by a one-metal ion mechanism. GIY-YIG homing endonucleases (GIY-HEs) are two-domain proteins with N-terminal GIY-YIG nuclease domains connected to C-terminal DNA-binding and they are thought to function as monomers. Using I-BmoI as a model GIY-HE, we test mechanisms by which the single active site is used to generate a double-strand break. We show that I-BmoI is partially disordered in the absence of substrate, and that the GIY-YIG domain alone has weak affinity for DNA. Significantly, we show that I-BmoI functions as a monomer at all steps of the reaction pathway and does not transiently dimerize or use sequential transesterification reactions to cleave substrate. Our results are consistent with the I-BmoI DNA-binding domain acting as a molecular anchor to tether the GIY-YIG domain to substrate, permitting rotation of the GIY-YIG domain to sequentially nick each DNA strand. These data highlight the mechanistic differences between monomeric GIY-HEs and dimeric or tetrameric GIY-YIG restriction enzymes, and they have implications for the use of the GIY-YIG domain in genome-editing applications.
authors.affiliation: Department of Biochemistry, Schulich School of Medicine and Dentistry, Western University, London, Ontario N6A 5C1, Canada.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Kleinstiver
authors.forename: Jason M
authors.initials: JM
authors.lastname: Wolfs
authors.forename: David R
authors.initials: DR
authors.lastname: Edgell
mesh_terms: D004247:DNA / Q000378:metabolism; D053903:DNA Breaks, Double-Stranded; D053904:DNA Breaks, Single-Stranded; D053837:DNA Cleavage; D004706:Endodeoxyribonucleases / Q000737:chemistry / Q000378:metabolism*; D017434:Protein Structure, Tertiary
chemical_list: D004247:DNA; D004706:Endodeoxyribonucleases; C432135:I-BmoI endonuclease
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