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. 2001 Oct 30;98(23):12990–12995. doi: 10.1073/pnas.241215698

Figure 4.

Figure 4

Characterization of DNA cleavage activity of N.AlwI. (A) N.AlwI cleavage activity on plasmid DNA. Plasmid DNA (≈200 ng) was incubated in a 15-μl digestion reaction for 20 min in the presence of AlwI (2 units), N.BstNBI (10 units), or N.AlwI (3 ng). Reactions were fractionated on a 1% agarose gel and stained with ethidium bromide. Lane 1, supercoiled plasmid pAC0; lane 2, pAC0 digested with AlwI; lane 3, pAC0 digested with N.AlwI; lane 4, pAC0 digested with N.BstNBI; lane 5, supercoiled plasmid pAC1; lane 6, pAC1 digested with AlwI; lane 7, pAC1 digested with N.AlwI; lane 8, pAC1 digested with N.BstNBI. S, supercoiled form of DNA; L, linearized form of DNA; N, nicked form of DNA. (B) N.AlwI cleavage activity on double-stranded oligodeoxyribonucleotides. Duplex substrates were prepared by annealing a fluorescein-labeled oligonucleotide with its nonlabeled complementary sequence (see Materials and Methods). Double-stranded duplex (≈50 pmol) was incubated with 100 ng of AlwI or 1 μg of N.AlwI in a 10:l reaction at 37°C for 1 h. Reactions were stopped by adding equal volume of a stop dye (95% formamide, 20 mM EDTA, 0.05% bromophenol blue, and 0.05% xylene cyanol FF). Approximately 1.4 μl of the reaction were separated on an 8% Quick Point sequencing gel (Invitrogen) and DNA bands were detected under a UV light. Detailed descriptions of lanes 1–8 are shown in C. (C) Schematic diagram of DNA substrates used in B. The pentamer (bases 24–28) sequence in the top strands indicates a wild-type recognition sequence (5′-GGATC-3′) or altered sequence (5′-GGATT-3′). Asterisks (*) indicate the fluorescein labels. (D) Mapping cleavage sites by modified sequencing reactions. pAC1 (4 μg), containing a single 5′-GGATC-3′ sequence, was used in manual sequencing reactions primed by either Alw15f (see Materials and Methods). Two additional reactions of each primer were extended fully without the addition of dideoxynucleotides and then digested with either AlwI (1 unit) or N.AlwI (1.4 ng). Reactions were fractionated on an 8% polyacrylamide gel and detected by autoradiography. (E) Identical to D except that the primer alw16r was used.