Figure 5.

Stalling the translocation of helicase motor stimulates cleavage. (A) Schematic representation of target DNA (100 bp) with PAM (TTC in red) end-labeled with 6-FAM at 5′ end of the non-target strand and biotinylated at 12th nucleotide (Target I). A similar target DNA without biotin (Target II) is also represented. Indicated in red triangle are the prominent cleavage sites. (B) Target (I) and (II) were incubated with streptavidin and Cascade/I-C to form interference complex. Cleavage was initiated by addition of Cas3 (0.2 and 0.5 μM) and 1 mM ATP/ADP/AMP-PNP. Prominent cleavages of the target (I) and (II) are indicated by a red triangle (∼60, 40 and 10 nt). The size of the DNA markers is shown in the lane M. A 20% denaturing PAGE was used to assess the cleavage of DNA.