title: Phosphorothioate substrates for the SfiI restriction endonuclease.
abstract: Oligodeoxynucleotides carrying the recognition sequence for the SfiI endonuclease were synthesised with phosphorothioates at the cleavage site. The Rp and Sp diastereoisomers of the oligonucleotides were separated by HPLC using a mobile phase containing L-cysteine. The duplex with Rp phosphorothioates was cleaved very slowly in the presence of Mg2+, though virtually complete cleavage was obtained with Mn2+. No significant cleavage of the duplex with Sp phosphorothioates occurred with either Mg2+ or Mn2+. When added to a plasmid with one SfiI site, the duplexes with either Rp or Sp phosphorothioates inhibited the rate at which SfiI cleaved the plasmid: a control duplex with oxyester linkages enhanced the rate of plasmid cleavage. In contrast to type IIe nucleases such as EcoRII and NaeI, which can be activated by non-hydrolysable analogues of their substrates, SfiI reactions require four susceptible phosphodiester bonds.
authors.affiliation: Department of Biochemistry, University of Bristol, UK.
authors.forename: T J
authors.initials: TJ
authors.lastname: Nobbs
authors.forename: S A
authors.initials: SA
authors.lastname: Williams
authors.forename: B A
authors.initials: BA
authors.lastname: Connolly
authors.forename: S E
authors.initials: SE
authors.lastname: Halford
mesh_terms: D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D008274:Magnesium; D008345:Manganese; D009838:Oligodeoxyribonucleotides / Q000378:metabolism; D017553:Phosphorus Compounds / Q000378:metabolism*; D013237:Stereoisomerism; D013379:Substrate Specificity; D013873:Thionucleotides / Q000378:metabolism*
chemical_list: D009838:Oligodeoxyribonucleotides; D017553:Phosphorus Compounds; D013873:Thionucleotides; D008345:Manganese; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium