Endonuclease specificity and sequence dependence of type IIS restriction enzymes

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 automat...

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Bibliographic Details
Published in:PloS one 2015-01, Vol.10 (1), p.e0117059-e0117059
Main Authors: Lundin, Sverker, Jemt, Anders, Terje-Hegge, Finn, Foam, Napoleon, Pettersson, Erik, Käller, Max, Wirta, Valtteri, Lexow, Preben, Lundeberg, Joakim
Format: Article
Language:English
Subjects:
Base Sequence
Cleavage
Deoxyribonucleases, Type II Site-Specific - genetics
Deoxyribonucleases, Type II Site-Specific - metabolism
Deoxyribonucleic acid
DNA
Endonuclease
Endonucleases - genetics
Endonucleases - metabolism
Enzymes
Gene expression
Genetic engineering
Genomes
Laboratories
Medicin och hälsovetenskap
Molecular biology
Nucleases
Sequences
Slippage
Substrate Specificity
Variation
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Summary: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.
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0117059