EMD-23845
Half integration complex of Cas4/Cas1/Cas2 and Cas4 still in the Non-PAM side
EMD-23845
Single-particle5.8 Å

Map released: 17/11/2021
Last modified: 29/05/2024
Sample Organism:
Geobacter sulfurreducens
Sample: Half integration complex of Cas4/Cas1/Cas2 and Cas4 still in the PAM side
Fitted models: 7mib (Avg. Q-score: 0.121)
Deposition Authors: Hu CY, Ke AK
Sample: Half integration complex of Cas4/Cas1/Cas2 and Cas4 still in the PAM side
Fitted models: 7mib (Avg. Q-score: 0.121)
Deposition Authors: Hu CY, Ke AK
Mechanism for Cas4-assisted directional spacer acquisition in CRISPR-Cas.
Hu C
,
Almendros C,
Nam KH
,
Costa AR
,
Vink JNA
,
Haagsma AC
,
Bagde SR
,
Brouns SJJ
,
Ke A
(2021) Nature , 598 , 515 - 520








(2021) Nature , 598 , 515 - 520
Abstract:
Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex2-4. 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)5,6 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.
Prokaryotes adapt to challenges from mobile genetic elements by integrating spacers derived from foreign DNA in the CRISPR array1. Spacer insertion is carried out by the Cas1-Cas2 integrase complex2-4. 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)5,6 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.