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Nucleic Acids Res. 2015 Dec 15;43(22):10821-30. doi: 10.1093/nar/gkv1213. Epub 2015 Nov 17.

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

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Faculty of Science, Department of Molecular Biology, University of Zagreb, Horvatovac 102a, Zagreb, Croatia.
School of Life Sciences, Queen's Medical Centre, University of Nottingham, NG72UH, UK.
School of Life Sciences, Queen's Medical Centre, University of Nottingham, NG72UH, UK


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.

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