A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity
Abstract
:1. Introduction
2. Results
2.1. Cas3 Nuclease Activity Is Modulated by Temperature Changes That Correspond with Conformational Re-Arrangement of Cas3 in Transition from 30 °C to 37 °C
2.2. A Single Tryptophan Residue (Trp-406) Modulates the Cas3 Nuclease Activity
2.3. Evidence That Trp-406 Is a ‘Gate’ in Cas3 That Controls Access of ssDNA to the Nuclease Active Site
2.4. Hyperactivity of Cas3W406A Nuclease Is Independent of Cas3 ATPase Activity and of the Cascade Complex
2.5. Cas3W406A Does Not Protect E. coli Cells from Phage Lysis
3. Discussion
4. Materials and Methods
4.1. E. coli Strains, Plasmids and Molecular Cloning
4.2. Protein Expression and Purification
4.3. DNA Substrate Preparation
4.4. Protein-Nucleic Acid Assays
4.5. ATPase Assays
4.6. Phage Sensitivity Assay by Plaque Formation
4.7. Construction of the Chromosomal Cas3 Point Mutant
4.8. Circular Dichroism Measurements and Analysis of Cas3 Secondary Structure
4.9. Molecular Dynamics Simulations of Cas3 Protein
Supplementary Materials
Author Contributions
Funding
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Secondary Structure | % Composition in Cas3 at 30 °C | % Composition in Cas3 at 35 °C | Change (Δ) |
---|---|---|---|
α-Helix | 18.1 | 14.3 | −3.8 |
β-antiparallel | 11.7 | 14.9 | 3.2 |
β-parallel | 0.0 | 0.0 | 0 |
Turn | 20.5 | 20.5 | 0 |
Other | 49.7 | 50.3 | 0.6 |
Secondary Structure | % Composition in Cas3W406A at 30 °C | % Composition in Cas3W406A at 35 °C | Change (Δ) |
---|---|---|---|
α-helix | 22.1 | 12.8 | −9.3 |
β-antiparallel | 22.0 | 22.8 | 0.8 |
β-parallel | 6.5 | 3.7 | −2.8 |
Turn | 10.3 | 11.7 | 1.4 |
Other | 39.1 | 49.0 | 9.9 |
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He, L.; Matošević, Z.J.; Mitić, D.; Markulin, D.; Killelea, T.; Matković, M.; Bertoša, B.; Ivančić-Baće, I.; Bolt, E.L. A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity. Int. J. Mol. Sci. 2021, 22, 2848. https://doi.org/10.3390/ijms22062848
He L, Matošević ZJ, Mitić D, Markulin D, Killelea T, Matković M, Bertoša B, Ivančić-Baće I, Bolt EL. A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity. International Journal of Molecular Sciences. 2021; 22(6):2848. https://doi.org/10.3390/ijms22062848
Chicago/Turabian StyleHe, Liu, Zoe Jelić Matošević, Damjan Mitić, Dora Markulin, Tom Killelea, Marija Matković, Branimir Bertoša, Ivana Ivančić-Baće, and Edward L. Bolt. 2021. "A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity" International Journal of Molecular Sciences 22, no. 6: 2848. https://doi.org/10.3390/ijms22062848