Uncropped gel—nuclease data from: A nicking class 1 OLD family nuclease encoded by Vibrio cholerae inhibits virbiophage replication and is countered by a direct inhibitor
Data files
Sep 08, 2025 version files 755 MB
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Figure_4_-_Replicate_A.tif
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Figure_4_-_Replicate_B.tif
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Figure_4_-_Replicate_C.tif
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Figure_S15A-_Replicate_A.tif
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Figure_S15A-_Replicate_B.tif
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Figure_S15A-_Replicate_C.tif
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Figure_S15C-_Replicate_A.tif
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Figure_S15C-_Replicate_B.tif
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Figure_S15C-_Replicate_C.tif
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Figure_S7A_-_Bacterial_gDNA_Replicate.tif
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Figure_S7A_-_Replicate_B.tif
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Figure_S7A_-_Replicate_C.tif
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Figure_S7A-_plasmid_-_Replicate_A-C.tif
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Figure_S7B_-_Replicate_A-C.tif
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Figure_S7C_-_Replicates_A_and_B.tif
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Figure_S7C_-_Replicates_C.tif
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Figure_S8-_Replicate_A.tif
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Figure_S8-_Replicate_B.tif
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Figure_S8-_Replicate_C.tif
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Figure_S9A-_Replicate_A.tif
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Figure_S9A-_Replicate_B.tif
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Figure_S9A-_Replicate_C.tif
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Figure_S9C-_Replicate_A.tif
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README.md
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Abstract
Bacteria are constantly threatened by their viral predators (phages), which has resulted in the development of defense systems for bacterial survival. One family of defense systems found widely across bacteria are OLD (for overcoming lysogenization defect) family nucleases. Despite recent discoveries regarding Class 2 and 4 OLD family nucleases and how phages overcome them, Class 1 OLD family nucleases warrant further study, as there has only been one anti-phage Class 1 OLD family nuclease described to date. Here, we identify the first bacteria-encoded Class 1 OLD family nuclease that exerts anti-phage activity: the Vibrio cholerae encoded Class 1 OLD family nuclease Vc OLD. We describe its disruption of the genome replication of the lytic vibriophage ICP1. Furthermore, we examine its in vitro activity, identifying Vc OLD as a DNA nickase. Finally, we identify the first direct inhibitor of a Class 1 OLD family nuclease, the ICP1-encoded Oad1. Our research further illuminates Class 1 OLD family nucleases’ role in phage defense and explores the dynamic arms race between V. cholerae and its predatory phage ICP1.
Dataset DOI: 10.5061/dryad.rxwdbrvnt
Description of the data and file structure
Files and variables
File: Figure_S15A-_Replicate_B.tif
Description: These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, the lanes are: Plasmid incubated with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM BSA, plasmid incubated with 100nM Vc OLD and 4nM BSA, plasmid incubated with 100nM Vc OLD and 100nM BSA, plasmid incubated with 100nM Vc OLD and 2500nM BSA, plasmid incubated with 100nM Vc OLD and 5000nM BSA, plasmid incubated with 5000nM BSA and without Vc OLD, 100nM Vc OLD incubated without plasmid, and 5000nM BSA incubated without plasmid. Replicate B.
File: Figure_S15A-_Replicate_C.tif
Description: These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, the lanes are: Plasmid incubated with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM BSA, plasmid incubated with 100nM Vc OLD and 4nM BSA, plasmid incubated with 100nM Vc OLD and 100nM BSA, plasmid incubated with 100nM Vc OLD and 2500nM BSA, plasmid incubated with 100nM Vc OLD and 5000nM BSA, plasmid incubated with 5000nM BSA and without Vc OLD, 100nM Vc OLD incubated without plasmid, and 5000nM BSA incubated without plasmid. Replicate C.
File: Figure_S15C-_Replicate_A.tif
Description: These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, the lanes are: Plasmid incubated with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM OAD1, plasmid incubated with 100nM Vc OLD and 4nM OAD1, plasmid incubated with 100nM Vc OLD and 100nM OAD1, plasmid incubated with 100nM Vc OLD and 2500nM OAD1, plasmid incubated with 100nM Vc OLD and 5000nM OAD1, plasmid incubated with 5000nM OAD1 and without Vc OLD, 100nM Vc OLD incubated without plasmid, and 5000nM OAD1 incubated without plasmid. Replicate A.
File: Figure_S15A-_Replicate_A.tif
Description: These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid incubated with BamHI-HF (NEB) , Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM BSA, plasmid incubated with 100nM Vc OLD and 4nM BSA, plasmid incubated with 100nM Vc OLD and 100nM BSA, plasmid incubated with 100nM Vc OLD and 2500nM BSA, plasmid incubated with 100nM Vc OLD and 5000nM BSA, plasmid incubated with 5000nM BSA and without Vc OLD, 100nM Vc OLD incubated without plasmid, and 5000nM BSA incubated without plasmid. Replicate A.
File: Figure_S15C-_Replicate_C.tif
Description: These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, the lanes are: Plasmid incubated with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM OAD1, plasmid incubated with 100nM Vc OLD and 4nM OAD1, plasmid incubated with 100nM Vc OLD and 100nM OAD1, plasmid incubated with 100nM Vc OLD and 2500nM OAD1, plasmid incubated with 100nM Vc OLD and 5000nM OAD1, plasmid incubated with 5000nM OAD1 and without Vc OLD, 100nM Vc OLD incubated without plasmid, and 5000nM OAD1 incubated without plasmid. Replicate C.
File: Figure_S15C-_Replicate_B.tif
Description: These data are linked to Supplemental Figure 15 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, the lanes are: Plasmid incubated with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 100nM Vc OLD and 2nM OAD1, plasmid incubated with 100nM Vc OLD and 4nM OAD1, plasmid incubated with 100nM Vc OLD and 100nM OAD1, plasmid incubated with 100nM Vc OLD and 2500nM OAD1, plasmid incubated with 100nM Vc OLD and 5000nM OAD1, plasmid incubated with 5000nM OAD1 and without Vc OLD, 100nM Vc OLD incubated without plasmid, and 5000nM OAD1 incubated without plasmid. Replicate B.
File: Figure_S9C-_Replicate_C.tif
Description: These data are linked to Supplemental Figure 9 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. For all of these rxns, the plasmid substrate contained 41 bp from the V. cholerae genome. From left to right the lanes are: a DNA ladder, Plasmid incubated with BamHI-HF (here the plasmid is not linearized due to the loss of the BamHI-HF cutting site), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate C.
File: Figure_S9A-_Replicate_A.tif
Description: These data are linked to Supplemental Figure 9 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. For all of these rxns, the plasmid substrate contained 41 bp from ICP1 genome. From left to right the lanes are: a DNA ladder, Plasmid incubated with BamHI-HF (here the plasmid is not linearized due to the loss of the BamHI-HF cutting site), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate A.
File: Figure_S9C-_Replicate_B.tif
Description: These data are linked to Supplemental Figure 9 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. For all of these rxns, the plasmid substrate contained 41 bp from the V. cholerae genome. From left to right the lanes are: a DNA ladder, Plasmid incubated with BamHI-HF (here the plasmid is not linearized due to the loss of the BamHI-HF cutting site), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate B.
File: Figure_S9A-_Replicate_C.tif
Description: These data are linked to Supplemental Figure 9 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. For all of these rxns, the plasmid substrate contained 41 bp from ICP1 genome. From left to right the lanes are: a DNA ladder, Plasmid incubated with BamHI-HF (here the plasmid is not linearized due to the loss of the BamHI-HF cutting site), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate C.
File: Figure_S9C-_Replicate_A.tif
Description: These data are linked to Supplemental Figure 9 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. For all of these rxns, the plasmid substrate contained 41 bp from the V. cholerae genome. From left to right the lanes are: a DNA ladder, Plasmid incubated with BamHI-HF (here the plasmid is not linearized due to the loss of the BamHI-HF cutting site), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate A.
File: Figure_S9A-_Replicate_B.tif
Description: These data are linked to Supplemental Figure 9 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. For all of these rxns, the plasmid substrate contained 41 bp from ICP1 genome. From left to right the lanes are: a DNA ladder, Plasmid incubated with BamHI-HF (here the plasmid is not linearized due to the loss of the BamHI-HF cutting site), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate B.
File: Figure_4_-_Replicate_C.tif
Description: These data are linked to Figure 4 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid linearized with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate C.
File: Figure_4_-_Replicate_B.tif
Description: These data are linked to Figure 4 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid linearized with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate B.
File: Figure_4_-_Replicate_A.tif
Description: These data are linked to Figure 4 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid linearized with BamHI-HF (NEB), Plasmid nicked with Nb.BtsI (NEB), plasmid not incubated with enzyme, plasmid incubated with 500nM Vc OLD, plasmid incubated with 250nM Vc OLD, plasmid incubated with 100nM Vc OLD, plasmid incubated with 50nM Vc OLD, plasmid incubated with 25nM Vc OLD, plasmid incubated with 10nM Vc OLD, and 500nM Vc OLD incubated without plasmid. Replicate A.
File: Figure_S8-_Replicate_B.tif
Description: These data are linked to Supplementary Figure 8 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid linearized with BamHI-HF (NEB) without ATP, Plasmid nicked with Nb.BtsI (NEB) without ATP, plasmid not incubated with enzyme or ATP, plasmid incubated with 100nM Vc OLD and 0.05mM ATP, plasmid incubated with 100nM Vc OLD and 0.1mM ATP, plasmid incubated with 100nM Vc OLD and and 0.2mM ATP, plasmid incubated with 100nM Vc OLD and and 0.5mM ATP, plasmid incubated with 100nM Vc OLD and and 1.0mM ATP, plasmid incubated without enzyme and with 1.0mM ATP 10nM Vc OLD, and 100nM Vc OLD incubated without plasmid or ATP. Replicate B.
File: Figure_S8-_Replicate_C.tif
Description: These data are linked to Supplementary Figure 8 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid linearized with BamHI-HF (NEB) without ATP, Plasmid nicked with Nb.BtsI (NEB) without ATP, plasmid not incubated with enzyme or ATP, plasmid incubated with 100nM Vc OLD and 0.05mM ATP, plasmid incubated with 100nM Vc OLD and 0.1mM ATP, plasmid incubated with 100nM Vc OLD and and 0.2mM ATP, plasmid incubated with 100nM Vc OLD and and 0.5mM ATP, plasmid incubated with 100nM Vc OLD and and 1.0mM ATP, plasmid incubated without enzyme and with 1.0mM ATP 10nM Vc OLD, and 100nM Vc OLD incubated without plasmid or ATP. Replicate C.
File: Figure_S8-_Replicate_A.tif
Description: These data are linked to Supplementary Figure 8 of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right the lanes are: Plasmid linearized with BamHI-HF (NEB) without ATP, Plasmid nicked with Nb.BtsI (NEB) without ATP, plasmid not incubated with enzyme or ATP, plasmid incubated with 100nM Vc OLD and 0.05mM ATP, plasmid incubated with 100nM Vc OLD and 0.1mM ATP, plasmid incubated with 100nM Vc OLD and and 0.2mM ATP, plasmid incubated with 100nM Vc OLD and and 0.5mM ATP, plasmid incubated with 100nM Vc OLD and and 1.0mM ATP, plasmid incubated without enzyme and with 1.0mM ATP 10nM Vc OLD, and 100nM Vc OLD incubated without plasmid or ATP. Replicate A.
File: Figure_S7C_-_Replicates_A_and_B.tif
Description: These data are linked to Supplementary Figure 7C of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, lanes contain: a DNA ladder, bacterial RNA incubated with RNAse, bacterial RNA not incubated with enzyme, bacterial RNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without bacterial RNA, a DNA ladder, bacterial RNA incubated with RNAse, bacterial RNA not incubated with enzyme, bacterial RNA incubated with 500nM Vc OLD, and 500nM Vc OLD incubated without bacterial RNA. Each five lanes represent an independent replicate.
File: Figure_S7A_-_Replicate_C.tif
Description: These data are linked to Supplementary Figure 7A of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. From left to right, lanes contain: a DNA ladder, phage gDNA incubated with DNAse, phage gDNA not incubated with enzyme, phage gDNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without gDNA, bacterial gDNA incubated with DNAse, bacterial gDNA not incubated with enzyme, bacterial gDNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without gDNA, phage gDNA incubated with DNAse, phage gDNA not incubated with enzyme, and phage gDNA incubated with 500nM Vc OLD. The lanes two through nine are the third replicates of the experiment. The final three lanes are the second replicate of this experiment with phage gDNA.
File: Figure_S7A-plasmid-_Replicate_A-C.tif
Description: These data are linked to Supplementary Figure 7A of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. Left to right, lanes contain: plasmid incubated with DNAse, plasmid gDNA not incubated with enzyme, plasmid gDNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without gDNA, plasmid incubated with DNAse, plasmid gDNA not incubated with enzyme, plasmid gDNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without gDNA, plasmid incubated with DNAse, plasmid gDNA not incubated with enzyme, plasmid gDNA incubated with 500nM Vc OLD, and 500nM Vc OLD incubated without gDNA. Each four lanes is a replicate of this experiment.
File: Figure_S7B_-_Replicate_A-C.tif
Description: These data are linked to Supplementary Figure 7B of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. Left to right, lanes contain: ssDNA incubated with DNAse, ssDNA not incubated with enzyme, ssDNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without ssDNA, ssDNA incubated without enzyme, ssDNA incubated with 500nM Vc OLD, ssDNA incubated with DNAse, 500nM Vc OLD incubated without ssDNA, ssDNA incubated with DNAse, ssDNA not incubated with enzyme, ssDNA incubated with 500nM Vc OLD, and 500nM Vc OLD incubated without ssDNA. Each four lanes is a replicate of this experiment.
File: Figure_S7A_-_Replicate_B.tif
Description: These data are linked to Supplementary Figure 7A of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. Red boxes indicates lanes not applicable to this study. For the lanes applicable to this study, left to right contain: phage gDNA incubated with DNAse, phage gDNA not incubated with enzyme, phage gDNA incubated with 500nM Vc OLD, 500nM Vc OLD incubated without gDNA, bacterial gDNA incubated with DNAse, bacterial gDNA not incubated with enzyme, bacterial gDNA incubated with 500nM Vc OLD, and 500nM Vc OLD incubated without gDNA. All of these are the second replicates of the experiment.
File: Figure_S7A_-_Bacterial_gDNA_Replicate.tif
Description: These data are linked to Supplementary Figure 7A of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. Red boxes indicates lanes not applicable to this study. For the lanes applicable to this study (middle), left to right contain: bacterial gDNA incubated with DNAse, bacterial gDNA not incubated with enzyme, bacterial gDNA incubated with 500nM Vc OLD, and 500nM Vc OLD incubated without gDNA.
File: Figure_S7C_-_Replicates_C.tif
Description: These data are linked to Supplementary Figure 7C of the manuscript. Uncropped image of a 0.8% agarose gel. 20uL of rxn was loaded per lane. The red box indicates lanes not applicable to this study. For lanes applicable, from left to right, lanes contain: a DNA ladder, bacterial RNA incubated with RNAse, bacterial RNA not incubated with enzyme, bacterial RNA incubated with 500nM Vc OLD, and 500nM Vc OLD incubated without bacterial RNA. This is the third replicate of this experiment.
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Nuclease assays with purified Vc OLD and Oad1 and bovine serum albumin (BSA) (Fisher BP1600) were carried out in 50-mM Tris–HCl, pH = 7.4, 50-mM NaCl, 10-mM MgCl2, 10-mM MnCl2, 10-mM CaCl2, 10NiCl2, and 10-mM ZnCl2. Frozen samples of all proteins were thawed and added to the buffer at varying concentrations. All nucleic acid substrates were eluted into H2O after nucleic acid collection. Double-stranded DNA (dsDNA) substrate was extracted from bacteria or phage following the procedure outlined in “Post-infection deep sequencing and coverage mapping.” Similarly, single-stranded DNA (ssDNA) (from M13 phage) was ordered from New England Biolabs (N4040S), extracted, and eluted into water. Total RNA was prepped from V. cholerae E7946 as described previously. Briefly, Bacterial strains were grown to an OD600 = 0.3, collected, and mixed 1:1 with ice-cold methanol. Methanol-treated samples were pelleted at 7000 × g at 4°C, and the resulting pellets were washed in ice-cold 1× phosphate-buffered saline. The washed pellets were resuspended in 200 μl TRI Reagent (Millipore/Sigma) and incubated for 5 min at room temperature. The samples were mixed with 40 μl chloroform, vortexed, and incubated for 10 min at room temperature. The chloroform-treated samples were then centrifuged at 12 000 × g for 10 min at 4°C. Following centrifugation, the upper (aqueous) phase was collected, promptly mixed with 110 μl 2-propanol and 11 μl pH 6.2 3-M sodium acetate, and mixed vigorously. The samples were centrifuged at 12 000 × g for 15 min at 4°C, and the pellets were washed twice with 75% ethanol. The washed pellets were incubated at 65°C for 3 min to evaporate residual ethanol. The RNA was resuspended in 10–30 μl of diethyl dicarbonate water, and RNA concentration were analyzed by NanoDrop. Plasmid DNA substrate, a pUC19 derivative, was prepped using the NEB Monarch Plasmid Miniprep Kit. All DNA substrates were added at 100 ng per 20 μl reaction. RNA was loaded at 500 ng per 20 μl reaction. Reactions containing DNA substrate were incubated at 37°C for 3 h, and then 1 μg/ml of Proteinase K was added to each reaction to digest bound protein and incubated at 37°C for 30 min. Reactions containing RNA substrate were incubated at 37°C for 30 min, and then 1 μg/ml of Proteinase K was added to each reaction to digest bound protein and incubated at 37°C for 30 min. To generate linear or nicked controls, 100 ng of vector was digested in 1× CutSmart Buffer for 1 h at 37°C with 10 U of BamHI-HF (NEB) or 10 U of Nb.BtsI (NEB) in 20 μl reactions, respectively, and then 1 μg/ml of Proteinase K was added to each reaction to digest bound protein and incubated at 37°C for 30 min. The entire reaction volume was loaded and run on a 0.8% agarose gel stained with GelRed.