Abstract
ABSTRACTCascade complexes underpin E. coli CRISPR-Cas immunity systems by stimulating ‘adaptation’ reactions that update immunity and by initiating ‘interference’ reactions that destroy invader DNA. Recognition of invader DNA in Cascade catalysed R-loops provokes DNA capture and its subsequent integration into CRISPR loci by Cas1 and Cas2. DNA capture processes are unclear but may involve RecG helicase, which stimulates adaptation during its role responding to genome instability. We show that Cascade is a potential source of genome instability because it blocks DNA replication and that RecG helicase alleviates this by dissociating Cascade. This highlights how integrating in vitro CRISPR-Cas interference and adaptation reactions with DNA replication and repair reactions will help to determine precise mechanisms underpinning prokaryotic adaptive immunity.
Highlights
CRISPR-Cas prokaryotic adaptive immunity protects cells from predation by phage and limits movement of mobile genetic elements (MGEs, e.g. plasmids) between cells (reviewed most recently in (Hille et al 2018)
Immunity is delivered when crRNA incorporated into CRISPR-Cas “Interference” complexes is targeted to MGE DNA leading to its destruction by nucleases
CRISPR-Cas immunity relies on insertion of spacer DNA into CRISPR loci
Summary
CRISPR-Cas prokaryotic adaptive immunity protects cells from predation by phage and limits movement of mobile genetic elements (MGEs, e.g. plasmids) between cells (reviewed most recently in (Hille et al 2018). We show that DNA replication, Cascade interference and DNA repair can be reconstituted in vitro as an integrated system and that RecG interacts with Cascade R-loop complexes and removes them.
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