Fig. 5.

Stereochemical course of DNA transesterification by BfiI. (A) Experimental strategy: the 25C/15(S)Rp duplex contains an R_p–PTO linkage at the site of bottom-strand DNA cleavage and a ³²P label [indicated by an asterisk (∗)] immediately upstream from the 3′-terminal nucleoside of the top strand (for nucleotide sequence, see Table 1). After incubation with BfiI to form hairpin DNA and subsequent purification (Fig. 2), aliquots were digested with either nuclease P1 or snake venom PDE. (B) Denaturing PAGE analysis of the nuclease P1 and the snake venom PDE digests of the hairpins formed from 25C/15(S)Rp and 25C/15(S)Sp (Table 1). The two substrates contain, respectively, R_p– and S_p–PTO linkages, as indicated above the gel. Lane M contains the mononucleotide marker dAMP.