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TitlePAM binding ensures orientational integration during Cas4-Cas1-Cas2-mediated CRISPR adaptation.
Journal, issue, pagesMol Cell, Vol. 82, Issue 22, Page 4353-44367.e6, Year 2022
Publish dateNov 17, 2022
AuthorsYukti Dhingra / Shravanti K Suresh / Puneet Juneja / Dipali G Sashital /
PubMed AbstractAdaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of ...Adaptation in CRISPR-Cas systems immunizes bacteria and archaea against mobile genetic elements. In many DNA-targeting systems, the Cas4-Cas1-Cas2 complex is required for selection and processing of DNA segments containing PAM sequences prior to integration of these "prespacer" substrates as spacers in the CRISPR array. We determined cryo-EM structures of the Cas4-Cas1-Cas2 adaptation complex from the type I-C system that encodes standalone Cas1 and Cas4 proteins. The structures reveal how Cas4 specifically reads out bases within the PAM sequence and how interactions with both Cas1 and Cas2 activate Cas4 endonuclease activity. The Cas4-PAM interaction ensures tight binding between the adaptation complex and the prespacer, significantly enhancing integration of the non-PAM end into the CRISPR array and ensuring correct spacer orientation. Corroborated with our biochemical results, Cas4-Cas1-Cas2 structures with substrates representing various stages of CRISPR adaptation reveal a temporally resolved mechanism for maturation and integration of functional spacers into the CRISPR array.
External linksMol Cell / PubMed:36272411 / PubMed Central
MethodsEM (single particle)
Resolution3.41 - 4.26 Å
Structure data

27159

8d3l

EMDB-27159, PDB-8d3l:
Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/PAM prespacer
Method: EM (single particle) / Resolution: 3.49 Å

27160

8d3m

EMDB-27160, PDB-8d3m:
Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/Processed prespacer
Method: EM (single particle) / Resolution: 3.41 Å

27161

8d3p

EMDB-27161, PDB-8d3p:
Type I-C Cas4-Cas1-Cas2 complex bound to half-site integration intermediate (HSI)
Method: EM (single particle) / Resolution: 4.26 Å

27162

8d3q

EMDB-27162, PDB-8d3q:
Type I-C Cas4-Cas1-Cas2 complex bound to a PAM/NoPAM prespacer
Method: EM (single particle) / Resolution: 3.9 Å

EMDB-27769: Type I-C Cas4-Cas1-Cas2 complex bound to a 24 bp duplex PAM/PAM prespacer
Method: EM (single particle) / Resolution: 3.9 Å

Chemicals

ChemComp-SF4:
IRON/SULFUR CLUSTER / Iron–sulfur cluster

ChemComp-MN:
Unknown entry

ChemComp-HOH:
WATER / Water

Source
  • alkalihalobacillus halodurans c-125 (bacteria)
  • synthetic construct (others)
KeywordsHYDROLASE/DNA / CRISPR Cas adaptation type I-C / HYDROLASE-DNA complex

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