engleski

Genome stability enzymes are essential for building CRISPR-Cas immunity in bacteria

CRISPR-Cas is a prokaryotic immune system built from capture and integration of invader DNA into CRISPR loci by Cas1 and Cas2 proteins, termed "Adaptation". In E. coli, Cascade-Cas3 degrades invader DNA to enact immunity, termed "Interference". Adaptation can be stimulated by interference (primed), or can be independent of interference (naive). We identified that host genome stability enzymes are required for adaptation ; primed adaptation requires RecG and PriA, naive adaptation requires RecB and both types require DNA polymerase I (PolA). Analysis of recG and priA phenotypes indicates interplay between primed adaptation , blocked replication fork and R-loop processing. We further show that Cas1 and Cas2 proteins specifically target substrates mimicking blocked forks. A model is proposed for DNA capture enabled by RecG, PriA and Cas3, or by RecB, according to different types of compromised DNA replication. PolA is proposed to synthesize DNA to fill single strand gaps during capture or integration.

CRISPR-Cas; RecG; PriA; PolA; E. coli

nije evidentirano