title: Indirect DNA Sequence Recognition and Its Impact on Nuclease Cleavage Activity.
abstract: LAGLIDADG meganucleases are DNA cleaving enzymes used for genome engineering. While their cleavage specificity can be altered using several protein engineering and selection strategies, their overall targetability is limited by highly specific indirect recognition of the central four base pairs within their recognition sites. In order to examine the physical basis of indirect sequence recognition and to expand the number of such nucleases available for genome engineering, we have determined the target sites, DNA-bound structures, and central four cleavage fidelities of nine related enzymes. Subsequent crystallographic analyses of a meganuclease bound to two noncleavable target sites, each containing a single inactivating base pair substitution at its center, indicates that a localized slip of the mutated base pair causes a small change in the DNA backbone conformation that results in a loss of metal occupancy at one binding site, eliminating cleavage activity.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA.
authors.forename: Abigail R
authors.initials: AR
authors.lastname: Lambert
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA.
authors.forename: Jazmine P
authors.initials: JP
authors.lastname: Hallinan
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA.
authors.forename: Betty W
authors.initials: BW
authors.lastname: Shen
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA.
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Chik
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA.
authors.forename: Jill M
authors.initials: JM
authors.lastname: Bolduc
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA.
authors.forename: Nadia
authors.initials: N
authors.lastname: Kulshina
authors.affiliation: Physical Sciences Division, School of STEM, University of Washington, 18115 Campus Way Northeast, Bothell, WA 98011, USA.
authors.forename: Lori I
authors.initials: LI
authors.lastname: Robins
authors.affiliation: Department of Biology, Seattle University, 901 12th Avenue, Seattle, WA 98122, USA.
authors.forename: Brett K
authors.initials: BK
authors.lastname: Kaiser
authors.affiliation: bluebird bio Inc. Suite 207, 1616 Eastlake Avenue East, Seattle, WA 98102, USA.
authors.forename: Jordan
authors.initials: J
authors.lastname: Jarjour
authors.affiliation: bluebird bio Inc. Suite 207, 1616 Eastlake Avenue East, Seattle, WA 98102, USA.
authors.forename: Kyle
authors.initials: K
authors.lastname: Havens
authors.affiliation: Seattle Children's Research Institute, 1900 Ninth Avenue, Seattle, WA 98101, USA.
authors.forename: Andrew M
authors.initials: AM
authors.lastname: Scharenberg
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, 1100 Fairview Avenue North, A3-025, Seattle, WA 98109, USA. Electronic address: bstoddar@fredhutch.org.
authors.forename: Barry L
authors.initials: BL
authors.lastname: Stoddard
mesh_terms: D001483:Base Sequence; D001665:Binding Sites; D004247:DNA / Q000737:chemistry* / Q000378:metabolism*; D053837:DNA Cleavage; D003851:Deoxyribonucleases / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D009690:Nucleic Acid Conformation; D011487:Protein Conformation; D013379:Substrate Specificity
chemical_list: D004247:DNA; D003851:Deoxyribonucleases