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Structure paper

TitleDnaQ mediates directional spacer acquisition in the CRISPR-Cas system by a time-dependent mechanism.
Journal, issue, pagesInnovation (Camb), Vol. 4, Issue 5, Page 100495, Year 2023
Publish dateSep 11, 2023
AuthorsDongmei Tang / Tingting Jia / Yongbo Luo / Biqin Mou / Jie Cheng / Shiqian Qi / Shaohua Yao / Zhaoming Su / Yamei Yu / Qiang Chen /
PubMed AbstractIn the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been ...In the spacer acquisition stage of CRISPR-Cas immunity, spacer orientation and protospacer adjacent motif (PAM) removal are two prerequisites for functional spacer integration. Cas4 has been implicated in both processing the prespacer and determining the spacer orientation. In Cas4-lacking systems, host 3'-5' DnaQ family exonucleases were recently reported to play a Cas4-like role. However, the molecular details of DnaQ functions remain elusive. Here, we characterized the spacer acquisition of the adaptation module of the type I-E system, in which a DnaQ domain naturally fuses with Cas2. We presented X-ray crystal structures and cryo-electron microscopy structures of this adaptation module. Our biochemical data showed that DnaQ trimmed PAM-containing and PAM-deficient overhangs with different efficiencies. Based on these results, we proposed a time-dependent model for DnaQ-mediated spacer acquisition to elucidate PAM removal and spacer orientation determination in Cas4-lacking CRISPR-Cas systems.
External linksInnovation (Camb) / PubMed:37663930 / PubMed Central
MethodsEM (single particle) / X-ray diffraction
Resolution1.448 - 4.12 Å
Structure data

34809

8hi1

EMDB-34809, PDB-8hi1:
Streptococcus thermophilus Cas1-Cas2- prespacer ternary complex
Method: EM (single particle) / Resolution: 3.09 Å

EMDB-34810: Streptococcus thermophilus Cas1-Cas2-DnaQ-prespacer complex
Method: EM (single particle) / Resolution: 3.92 Å

EMDB-34811: Streptococcus thermophilus Cas1-Cas2-DnaQ-prespacer complex, conformation2
Method: EM (single particle) / Resolution: 4.12 Å

PDB-8h18:
Crystal structure of DnaQ domain of Streptococcus thermophilus strain DGCC 7710
Method: X-RAY DIFFRACTION / Resolution: 1.499 Å

PDB-8h2f:
Crystal structure of DnaQ domain in complex witn TMP of Streptococcus thermophilus strain DGCC 7710
Method: X-RAY DIFFRACTION / Resolution: 1.448 Å

Chemicals

ChemComp-GOL:
GLYCEROL

ChemComp-MG:
Unknown entry

ChemComp-HOH:
WATER

ChemComp-TMP:
THYMIDINE-5'-PHOSPHATE

Source
  • streptococcus thermophilus dgcc 7710 (bacteria)
  • phage #d (virus)
  • Streptococcus thermophilus (bacteria)
KeywordsLYASE / nuclease / IMMUNE SYSTEM / CRISPR / cas / adaptation

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