Structural basis for RNA-guided DNA degradation by Cas5-HNH/Cascade complex

Area of Science:

• Molecular Biology
• Structural Biology
• Microbiology

Background:

• The Type I-E CRISPR-Cas system provides prokaryotic defense against foreign DNA.
• A novel HNH-comprising Cascade complex offers precise DNA cleavage, distinct from the typical Cas3 nuclease.
• Understanding this system's structure and function is crucial for its biotechnological applications.

Purpose of the Study:

• To elucidate the near-atomic structures of the Cas5-HNH/Cascade complex from Candidatus Cloacimonetes bacterium.
• To investigate the structural basis for the complex's DNA binding, activation, and regulation.
• To provide insights into the assembly and enzymatic activity of this unique CRISPR-Cas nuclease.

Main Methods:

• Near-atomic cryo-electron microscopy (cryo-EM) was employed to determine the structures of the Cas5-HNH/Cascade complex.
• Biochemical assays were performed to assess the impact of mutations and divalent ions on enzymatic activity.
• Structural analysis focused on subunit interactions and conformational changes upon DNA binding.

Main Results:

• Cryo-EM structures revealed extensive interactions between the Cas5-HNH domain and subunits Cas6 and Cas11.
• Mutations in these interaction sites significantly reduced the complex's nuclease activity.
• DNA binding induced a compact conformation, activating the Cas5-HNH/Cascade complex.
• Divalent metal ions (Zn2+, Co2+, Ni2+) were found to inhibit activity by destabilizing the complex.

Conclusions:

• The study provides detailed structural insights into the assembly and activation mechanism of the Cas5-HNH/Cascade complex.
• The findings highlight the role of specific subunit interactions in mediating enzymatic function.
• The regulatory role of divalent ions offers potential avenues for controlling CRISPR-Cas activity.