A moonlighting nuclease puts CRISPR in its place

Area of Science:

• Microbiology
• Molecular Biology
• Genetics

Background:

• CRISPR-Cas systems provide adaptive immunity in prokaryotes by integrating foreign DNA spacers into CRISPR loci.
• Spacer integration is primarily mediated by the Cas1/Cas2 integrase complex, often requiring the accessory Cas4 exonuclease.
• The function of Cas4 or its functional equivalent in CRISPR-Cas systems lacking Cas4 remains largely unknown.

Purpose of the Study:

• To investigate the mechanism of spacer integration in CRISPR-Cas systems that lack the canonical Cas4 exonuclease.
• To identify the protein(s) responsible for Cas4-like activity in subtype I-E CRISPR-Cas systems.

Main Methods:

• Comparative genomic analysis of CRISPR-Cas systems.
• Biochemical assays to test exonuclease activity.
• In vitro reconstitution of CRISPR-Cas integration reactions.

Main Results:

• Subtype I-E CRISPR-Cas systems, which lack Cas4, possess Cas4-like exonuclease activity.
• This activity is supplied by DnaQ-superfamily exonucleases, enzymes with known roles in DNA replication fidelity.
• DnaQ-superfamily exonucleases are recruited to the CRISPR locus to facilitate spacer integration.

Conclusions:

• DnaQ-superfamily exonucleases play a dual role, acting as both DNA repair enzymes and essential components of CRISPR-Cas adaptive immunity.
• This finding highlights the phenomenon of 'cellular machinery moonlighting', where proteins acquire new functions.
• The study elucidates a key step in CRISPR-Cas adaptation, particularly in systems lacking Cas4.