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. 2020 Jan 25;48(5):2486–2501. doi: 10.1093/nar/gkz1218

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© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com

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Figure 6. — Roadblock in the translocation of Cas3/I-C stimulates cleavage (A). (D) A schematic representation of 60 nt 5′ 6-FAM labeled ssDNA with biotin at 12th (Target A) and 20th nucleotide (Target B), respectively is shown. (B) and (E) ssDNA mentioned above was incubated with 200 nM of Cas3/I-C for several time points, and anisotropy measurements were recorded. With time, the decrease in anisotropy values was observed for ssDNA with a biotin roadblock. (C) and (F) ssDNA was pre-incubated with streptavidin before the addition of Cas3/I-C. Prominent DNA cleavage products were observed in the presence of ATP at ∼40 nt in target A and ∼50 nt in target B, indicated with a red triangle. In the presence of AMP-PNP, higher Cas3 concentration was required for the cleavage. The size of the DNA markers is shown in the lane M. A 20% denaturing PAGE was used to assess the cleavage of DNA.