Fig. 1.

E. coli CRISPR system and nuclease activity of YgbT. (A) CRISPR-Cas system of E. coli K12 W3110. Repeats are shown as yellow diamonds, and spacers as colored rectangular boxes. (B, C, D, E) Cleavage of ssDNA (B), ssRNA (C), dsDNA (D) or dsRNA (E) by YgbT (denaturing PAGE/autoradiography). The 5′-[³²P]-labeled ssDNA (34 nt), ssRNA (37 nt) or in a complex with complementary oligonucleotides was incubated without protein (C), with YgbT for the indicated times (37 oC) or in the presence of 10 mM EDTA for 60 min (EDTA). (F) Cleavage of HJ1 by YgbT (native PAGE/autoradiography). The 5′-[³²P]-labeled (*) HJ1 substrate was incubated at 37 oC for 45 min without protein (C) or with YgbT (1) or RuvC (2). (G) Dependence of HJ1 cleavage on YgbT concentration (native PAGE). The 5′-[³²P]-labeled HJ1 substrate (20 nM) was incubated in the absence (1) or in the presence of YgbT (Lanes 2–8: 0.05, 0.1, 0.2, 0.4, 0.8, or 1.0 μg) at 37 oC for 30 min. (H) Effect of divalent metal cations on HJ1 cleavage by YgbT (30 min, 37 °C, 5 mM metals or 10 mM EDTA; native PAGE). (I, J) Denaturing gel analysis of the cleavage of HJ1 by YgbT (triangles) or RuvC (arrows). The HJ1 substrate labeled by [³²P] on different strands (A, B, C, or D) was incubated without protein (C) or with YgbT (1) or RuvC (2). (K) Re-ligation assay of the HJ cleavage products generated by YgbT or RuvC. The [³²P]-labeled asymmetric HJ2 was incubated (30 min at 37oC) without protein (C) or with RuvC or YgbT, and the reaction products were then incubated with T4 DNA ligase prior to PAGE. Re-ligation products are indicated by arrows.