A tryptophan ‘gate’ in the CRISPR-Cas3 nuclease controls ssDNA entry into the nuclease site, that when removed results in nuclease hyperactivity
| Autor: | Zoe Jelić Matošević, Branimir Bertoša, Liu He, Marija Matković, Dora Markulin, Ivana Ivančić-Baće, Damjan Mitić, Edward L. Bolt, Tom Killelea |
|---|---|
| Jazyk: | chorvatština |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Protein Conformation CRISPR-Associated Proteins lcsh:Chemistry chemistry.chemical_compound Adenosine Triphosphate Protein structure Catalytic Domain Cas3 CRISPR lcsh:QH301-705.5 Spectroscopy Gene Editing Alanine biology Effector Circular Dichroism Escherichia coli Proteins Temperature Tryptophan General Medicine Computer Science Applications helicase Chemistry genome editing 030106 microbiology Allosteric regulation Mutation Missense DNA Single-Stranded Article Catalysis Inorganic Chemistry 03 medical and health sciences Escherichia coli Humans Amino Acid Sequence Physical and Theoretical Chemistry Molecular Biology Biology Nuclease Sequence Homology Amino Acid Organic Chemistry DNA Helicases Helicase Active site DNA 030104 developmental biology lcsh:Biology (General) lcsh:QD1-999 chemistry Biophysics biology.protein CRISPR-Cas Systems |
| Zdroj: | International journal of molecular sciences International Journal of Molecular Sciences Volume 22 Issue 6 International Journal of Molecular Sciences, Vol 22, Iss 2848, p 2848 (2021) |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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