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-Structure paper
タイトル | Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas. |
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ジャーナル・号・ページ | Nature, Vol. 598, Issue 7881, Page 515-520, Year 2021 |
掲載日 | 2021年9月29日 |
著者 | Chunyi Hu / Cristóbal Almendros / Ki Hyun Nam / Ana Rita Costa / Jochem N A Vink / Anna C Haagsma / Saket R Bagde / Stan J J Brouns / Ailong Ke / |
PubMed 要旨 | Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A ...Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array. Spacer insertion is carried out by the Cas1-Cas2 integrase complex. A substantial fraction of CRISPR-Cas systems use a Fe-S cluster containing Cas4 nuclease to ensure that spacers are acquired from DNA flanked by a protospacer adjacent motif (PAM) and inserted into the CRISPR array unidirectionally, so that the transcribed CRISPR RNA can guide target searching in a PAM-dependent manner. Here we provide a high-resolution mechanistic explanation for the Cas4-assisted PAM selection, spacer biogenesis and directional integration by type I-G CRISPR in Geobacter sulfurreducens, in which Cas4 is naturally fused with Cas1, forming Cas4/Cas1. During biogenesis, only DNA duplexes possessing a PAM-embedded 3'-overhang trigger Cas4/Cas1-Cas2 assembly. During this process, the PAM overhang is specifically recognized and sequestered, but is not cleaved by Cas4. This 'molecular constipation' prevents the PAM-side prespacer from participating in integration. Lacking such sequestration, the non-PAM overhang is trimmed by host nucleases and integrated to the leader-side CRISPR repeat. Half-integration subsequently triggers PAM cleavage and Cas4 dissociation, allowing spacer-side integration. Overall, the intricate molecular interaction between Cas4 and Cas1-Cas2 selects PAM-containing prespacers for integration and couples the timing of PAM processing with the stepwise integration to establish directionality. |
リンク | Nature / PubMed:34588691 / PubMed Central |
手法 | EM (単粒子) |
解像度 | 3.2 - 5.8 Å |
構造データ | EMDB-23839, PDB-7mi4: EMDB-23840, PDB-7mi5: EMDB-23843, PDB-7mi9: EMDB-23845: Half integration complex of Cas4/Cas1/Cas2 and Cas4 still in the Non-PAM side EMDB-23847, PDB-7mid: EMDB-23849: |
化合物 | ChemComp-SF4: ChemComp-MN: |
由来 |
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キーワード | HYDROLASE/DNA / CRISPR/Cas / Cas4 / PAM recognition / HYDROLASE-DNA complex / full integration / half integration |