PHOSPHOROTHIOATE SUBSTRATES FOR THE SFII RESTRICTION-ENDONUCLEASE

Oligodeoxynucleotides carrying the recognition sequence for the Sfil e ndonuclease were synthesised with phosphorothioates at the cleavage si te. The R-p and S-p diastereoisomers of the oligonucleotides were sepa rated by HPLC using a mobile phase containing L-cysteine. The duplex w ith R-p phosphorothioates was cleaved very slowly in the presence of M g2+, though virtually complete cleavage was obtained with Mn2+. No sig nificant cleavage of the duplex with S-p phosphorothioates occurred wi th either Mg2+ or Mn2+. When added to a plasmid with one Sfil site, th e duplexes with either R-p or S-p phosphorothioates inhibited the rate at which Sfil cleaved the plasmid: a control duplex with oxyester lin kages enhanced the rate of plasmid cleavage. In contrast to type lle n ucleases such as EcoRII and Nael, which can be activated by non-hydrol ysable analogues of their substrates, Sfil reactions require four susc eptible phosphodiester bonds.