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Article

A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity

1
School of Life Sciences, University of Nottingham, Nottingham NG7 2UH, UK
2
Department of Chemistry, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia
3
Department of Biology, Faculty of Science, University of Zagreb, 10000 Zagreb, Croatia
4
Institute Ruđer Bošković, 10000 Zagreb, Croatia
*
Authors to whom correspondence should be addressed.
Academic Editor: Yves Henry
Int. J. Mol. Sci. 2021, 22(6), 2848; https://doi.org/10.3390/ijms22062848
Received: 27 January 2021 / Revised: 5 March 2021 / Accepted: 8 March 2021 / Published: 11 March 2021
Cas3 is a ssDNA-targeting nuclease-helicase essential for class 1 prokaryotic CRISPR immunity systems, which has been utilized for genome editing in human cells. Cas3-DNA crystal structures show that ssDNA follows a pathway from helicase domains into a HD-nuclease active site, requiring protein conformational flexibility during DNA translocation. In genetic studies, we had noted that the efficacy of Cas3 in CRISPR immunity was drastically reduced when temperature was increased from 30 °C to 37 °C, caused by an unknown mechanism. Here, using E. coli Cas3 proteins, we show that reduced nuclease activity at higher temperature corresponds with measurable changes in protein structure. This effect of temperature on Cas3 was alleviated by changing a single highly conserved tryptophan residue (Trp-406) into an alanine. This Cas3W406A protein is a hyperactive nuclease that functions independently from temperature and from the interference effector module Cascade. Trp-406 is situated at the interface of Cas3 HD and RecA1 domains that is important for maneuvering DNA into the nuclease active site. Molecular dynamics simulations based on the experimental data showed temperature-induced changes in positioning of Trp-406 that either blocked or cleared the ssDNA pathway. We propose that Trp-406 forms a ‘gate’ for controlling Cas3 nuclease activity via access of ssDNA to the nuclease active site. The effect of temperature in these experiments may indicate allosteric control of Cas3 nuclease activity caused by changes in protein conformations. The hyperactive Cas3W406A protein may offer improved Cas3-based genetic editing in human cells. View Full-Text
Keywords: helicase; Cas3; CRISPR; genome editing helicase; Cas3; CRISPR; genome editing
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MDPI and ACS Style

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

AMA Style

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 Style

He, Liu; Matošević, Zoe J.; Mitić, Damjan; Markulin, Dora; Killelea, Tom; Matković, Marija; Bertoša, Branimir; Ivančić-Baće, Ivana; Bolt, Edward L. 2021. "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. 22, no. 6: 2848. https://doi.org/10.3390/ijms22062848

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