Figure 1.

Cas3/I-C cleaves the target DNA indiscriminately. (A) A schematic overview of type I-C CRISPR-Cas locus from B. halodurans C-125. Genes are drawn to represent locus approximately to the scale. Effector nuclease (cas3), cascade (cas5, cas7 and cas8) and adaptation (cas1, cas2 and cas4) genes are shown. Repeats are shown as diamonds in magenta and spacers as rectangles in different colors. (B) ssDNA and dsDNA represent single-stranded and double-stranded DNA substrates, respectively. Cas3/I-C cleaves both single (M13mp18) and double (pQE2) stranded DNA in the presence of divalent metal ion (Mg²⁺) and ATP. DNA marker (M) positions are shown on the left. (C) Methylated and unmethylated DNA fragments are of the same size (2.5 kb). Unmethylated DNA was generated using Pfu polymerase that generates blunt-end products. The methylated DNA fragment of 2.5 kb was released from plasmid using restriction enzymes NdeI and KpnI. An additional band of 4.8 kb corresponds to the linearized vector pQE2. DNase activity is not sensitive to methylation. The results were visualized using 0.8% agarose gel electrophoresis. Dotted line indicates a discontinuity in the gel for clarity and M denotes the position of DNA marker. (D andE) DNase/RNase activity depends on the size of the substrate. DNase and RNase activities were assessed on 0.8% agarose and 12% denaturing PAGE, respectively. To test the nuclease activity, 500 nM Cas3/I-C was incubated with 100 nM of 2.5 kb or 400 bp DNA. Similarly, 1.5 μM Cas3/I-C was used against 300 nM of 70 bp DNA. RNA concentration was 50 nM. Cas3/I-C preferred large DNA substrates (2.5 kb), whereas smaller DNA (400 and 70 bp) and RNA (150 nt) substrates were not completely cleaved even after 60 min of incubation. M denotes the position of DNA marker. (F andG) DNA unwinding was tested using fluorescently labeled partial DNA duplex. Displacement of 36 nt was observed when a substrate containing 3′ overhang was used. About 15% native PAGE was used to analyze the results.