Different genome stability proteins underpin primed and naïve adaptation in E. coli CRISPR-Cas immunity.

Ivana Ivančić-Baće,Stephen J Wearne,Edward L Bolt,Simon D Cass

doi:10.1093/nar/gkv1213

Abstract

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) loci, termed ‘Adaptation’, which is dependent on Cas1 and Cas2 proteins. In Escherichia coli, Cascade-Cas3 degrades invader DNA to effect immunity, termed ‘Interference’. Adaptation can interact with interference (‘primed’), or is independent of it (‘naïve’). We demonstrate that primed adaptation requires the RecG helicase and PriA protein to be present. Genetic analysis of mutant phenotypes suggests that RecG is needed to dissipate R-loops at blocked replication forks. Additionally, we identify that DNA polymerase I is important for both primed and naive adaptation, and that RecB is needed for naïve adaptation. Purified Cas1-Cas2 protein shows specificity for binding to and nicking forked DNA within single strand gaps, and collapsing forks into DNA duplexes. The data suggest that different genome stability systems interact with primed or naïve adaptation when responding to blocked or collapsed invader DNA replication. In this model, RecG and Cas3 proteins respond to invader DNA replication forks that are blocked by Cascade interference, enabling DNA capture. RecBCD targets DNA ends at collapsed forks, enabling DNA capture without interference. DNA polymerase I is proposed to fill DNA gaps during spacer integration.

Highlights

CRISPR-Cas are adaptive immune systems in prokaryotes that act against invasive genetic elements [1]
We investigated adaptation in E. coli strains deleted for genes encoding proteins that help to maintain genome stability by DNA repair and homologous recombination
We present new insights into how genome stability systems underpin the building of CRISPR-Cas immunity by either primed or naıve adaptation in E. coli

Summary

Introduction

CRISPR-Cas are adaptive immune systems in prokaryotes that act against invasive genetic elements (e.g. phages and plasmids) [1]. Cas (CRISPR-associated) proteins catalytically process CRISPR DNA and RNA to bring about immunity through targeting and destruction of invader nucleic acids. Building of the CRISPR immune system requires Cas and Cas proteins, and is likely to occur in two major events; capture of DNA fragments (‘protospacers’) from an invader, and integration of protospacers into a CRISPR locus as a new spacer. Spacer integration is accompanied by synthesis of a new repeat by an unknown factor. These processes are called CRISPR ‘Adaptation’ or ‘Acquisition’ [1,4,5,6,7,8,9]

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Conclusion