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Pregled bibliografske jedinice broj: 1119578

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


He, Liu; Jelić Matošević, Zoe; Mitić, Damjan; Markulin, Dora; Killelea, Tom; Matković, Marija; Bertoša, Branimir; Ivančić-Baće, Ivana; Bolt, Edward L.
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 (2021), 6; 1, 19 doi:10.3390/ijms22062848 (međunarodna recenzija, članak, znanstveni)


CROSBI ID: 1119578 Za ispravke kontaktirajte CROSBI podršku putem web obrasca

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

Autori
He, Liu ; Jelić Matošević, Zoe ; Mitić, Damjan ; Markulin, Dora ; Killelea, Tom ; Matković, Marija ; Bertoša, Branimir ; Ivančić-Baće, Ivana ; Bolt, Edward L.

Izvornik
International journal of molecular sciences (1422-0067) 22 (2021), 6; 1, 19

Vrsta, podvrsta i kategorija rada
Radovi u časopisima, članak, znanstveni

Ključne riječi
helicase ; Cas3 ; CRISPR ; genome editing

Sažetak
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.

Izvorni jezik
Engleski

Znanstvena područja
Kemija, Biologija



POVEZANOST RADA


Projekti:
HRZZ-IP-2016-06-8861 - Cas3 kao kontrolna točka obrane CRISPR-Cas: razjašnjenje njegove regulacije istraživanjem stabilnosti proteina i prepisivanja u bakteriji Escherichia coli (Cas3 status) (Ivančić Baće, Ivana, HRZZ - 2016-06) ( POIROT)

Ustanove:
Institut "Ruđer Bošković", Zagreb,
Prirodoslovno-matematički fakultet, Zagreb

Citiraj ovu publikaciju

He, Liu; Jelić Matošević, Zoe; Mitić, Damjan; Markulin, Dora; Killelea, Tom; Matković, Marija; Bertoša, Branimir; Ivančić-Baće, Ivana; Bolt, Edward L.
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 (2021), 6; 1, 19 doi:10.3390/ijms22062848 (međunarodna recenzija, članak, znanstveni)
He, L., Jelić Matošević, Z., Mitić, D., Markulin, D., Killelea, T., Matković, M., Bertoša, B., Ivančić-Baće, I. & Bolt, E. (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 (6), 1, 19 doi:10.3390/ijms22062848.
@article{article, year = {2021}, pages = {19}, DOI = {10.3390/ijms22062848}, chapter = {1}, keywords = {helicase, Cas3, CRISPR, genome editing}, journal = {International journal of molecular sciences}, doi = {10.3390/ijms22062848}, volume = {22}, number = {6}, issn = {1422-0067}, title = {A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity}, keyword = {helicase, Cas3, CRISPR, genome editing}, chapternumber = {1} }
@article{article, year = {2021}, pages = {19}, DOI = {10.3390/ijms22062848}, chapter = {1}, keywords = {helicase, Cas3, CRISPR, genome editing}, journal = {International journal of molecular sciences}, doi = {10.3390/ijms22062848}, volume = {22}, number = {6}, issn = {1422-0067}, title = {A Tryptophan ‘Gate’ in the CRISPR-Cas3 Nuclease Controls ssDNA Entry into the Nuclease Site, That When Removed Results in Nuclease Hyperactivity}, keyword = {helicase, Cas3, CRISPR, genome editing}, chapternumber = {1} }

Časopis indeksira:


  • Current Contents Connect (CCC)
  • Web of Science Core Collection (WoSCC)
    • Science Citation Index Expanded (SCI-EXP)
    • SCI-EXP, SSCI i/ili A&HCI
  • Scopus
  • MEDLINE


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