title: Endonuclease specificity and sequence dependence of type IIS restriction enzymes.
abstract: Restriction enzymes that recognize specific sequences but cleave unknown sequence outside the recognition site are extensively utilized tools in molecular biology. Despite this, systematic functional categorization of cleavage performance has largely been lacking. We established a simple and automatable model system to assay cleavage distance variation (termed slippage) and the sequence dependence thereof. We coupled this to massively parallel sequencing in order to provide sensitive and accurate measurement. With this system 14 enzymes were assayed (AcuI, BbvI, BpmI, BpuEI, BseRI, BsgI, Eco57I, Eco57MI, EcoP15I, FauI, FokI, GsuI, MmeI and SmuI). We report significant variation of slippage ranging from 1-54%, variations in sequence context dependence, as well as variation between isoschizomers. We believe this largely overlooked property of enzymes with shifted cleavage would benefit from further large scale classification and engineering efforts seeking to improve performance. The gained insights of in-vitro performance may also aid the in-vivo understanding of these enzymes.
authors: Science for Life Laboratory, KTH, Gene Technology, Solna, 171 65, Sweden.
authors: Sverker
authors: S
authors: Lundin
authors: Science for Life Laboratory, KTH, Gene Technology, Solna, 171 65, Sweden.
authors: Anders
authors: A
authors: Jemt
authors: LingVitae AS, Husøysund, 3132, Norway.
authors: Finn
authors: F
authors: Terje-Hegge
authors: LingVitae AS, Husøysund, 3132, Norway.
authors: Napoleon
authors: N
authors: Foam
authors: LingVitae AS, Husøysund, 3132, Norway.
authors: Erik
authors: E
authors: Pettersson
authors: LingVitae AS, Husøysund, 3132, Norway.
authors: Max
authors: M
authors: Käller
authors: LingVitae AS, Husøysund, 3132, Norway.
authors: Valtteri
authors: V
authors: Wirta
authors: LingVitae AS, Husøysund, 3132, Norway.
authors: Preben
authors: P
authors: Lexow
authors: Science for Life Laboratory, KTH, Gene Technology, Solna, 171 65, Sweden.
authors: Joakim
authors: J
authors: Lundeberg
mesh_terms: D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific / Q000235:genetics* / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D013379:Substrate Specificity
chemical_list: D004720:Endonucleases; D015252:Deoxyribonucleases, Type II Site-Specific
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