Phosphorothioate substrates for the SfiI restriction endonuclease.
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.
Nobbs
T J
TJ
Department of Biochemistry, University of Bristol, UK.
Williams
S A
SA
Connolly
B A
BA
Halford
S E
SE
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*
D009838:Oligodeoxyribonucleotides; D017553:Phosphorus Compounds; D013873:Thionucleotides; D008345:Manganese; C041749:endodeoxyribonuclease SfiI; D015252:Deoxyribonucleases, Type II Site-Specific; D008274:Magnesium