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

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

Authors

Ivana Ivančić-Baće

Simon Cass

Stephen J. Wearne

Edward L. Bolt ed.bolt@nottingham.ac.uk

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.

Journal Article Type Article
Publication Date Nov 17, 2015
Journal Nucleic Acids Research
Print ISSN 0305-1048
Electronic ISSN 1362-4962
Publisher Oxford University Press (OUP)
Peer Reviewed Peer Reviewed
DOI https://doi.org/10.1093/nar/gkv1213
Publisher URL http://nar.oxfordjournals.org/content/43/22/10821.short
Copyright Statement Copyright information regarding this work can be found at the following address: http://creativecommons.org/licenses/by/4.0

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