This study presents cryo-electron microscopy structures of the type I-E Cascade and provides insights into PAM-dependent R-loop formation, Cas3 recruitment and substrate handover.
In type I CRISPR–Cas systems, foreign DNA is detected by the CRISPR-associated complex for antiviral defence (Cascade). This surveillance complex contains Cas proteins and CRISPR RNA (crRNA), which recognize DNA target sequences (protospacers) that are flanked by an optimal protospacer-adjacent motif (PAM). During the target-searching process, crRNA binds to the complementary target DNA strand to form a seed bubble, whereas the non-targeting strand is displaced (R-loop formation). Subsequently, the nuclease–helicase enzyme Cas3 is recruited to degrade the non-target and target DNA strands.
mechanistic insights into PAM-dependent R-loop formation, Cas3 recruitment and substrate handover
This study presents cryo-electron microscopy structures of the type I-E Cascade from Thermobifida fusca, which forms a seed sequence bubble and a full R-loop structure, at 3.8 Å and 3.3 Å resolution, respectively, thus capturing the early events in R-loop formation. These structural snapshots revealed that PAM recognition is coupled to the bending of the target DNA and spontaneous DNA unwinding, which leads to the establishment of a seed bubble as the DNA–crRNA heteroduplex forms. Further unwinding of the entire protospacer leads to the completion of R-loop formation, which triggers a conformational change in Cascade. Moreover, the degradation factor Cas3 was shown to specifically bind to Cascade once R-loop formation was completed, which suggests a regulatory mechanism to prevent premature recruitment of Cas3 until the entire protospacer region has been sequence validated by the crRNA guide. Finally, Cascade was shown to create a bulge in the non-target DNA strand that facilitates the exchange of the non-target DNA strand from Cascade to Cas3 for degradation. Collectively, the structures provide mechanistic insights into PAM-dependent R-loop formation, Cas3 recruitment and substrate handover.
References
Xiao, Y., Luo, M. et al. Structure basis for directional R-loop formation and substrate handover mechanisms in type I CRISPR–Cas system. Cell https://doi.org/10.1016/j.cell.2017.06.012. 2017
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Du Toit, A. Cascade keeps its targets in the loop. Nat Rev Microbiol 15, 450 (2017). https://doi.org/10.1038/nrmicro.2017.80
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DOI: https://doi.org/10.1038/nrmicro.2017.80