engleski

Interplay of CRISPR adaptation, recombination and host nucleases in Escherichia coli

CRISPR-Cas system is a prokaryotic adaptive immune system against foreign genetic elements. Immunity is acquired through insertion of small fragments (spacers) of invader DNA into a CRISPR array. This process is called adaptation, and can be mediated solely by the protein complex Cas1-Cas2 (“naïve adaptation”). In E. coli, RecBCD is thought to aid naïve adaptation by generating single-stranded DNA intermediates that are reannealed and further processed by Cas1-Cas2, and then integrated into the CRISPR array. In this work, we wanted to better understand the role of RecBCD and other host exonucleases in the process of prespacer preparation. Our genetic analysis shows that nuclease activity of RecBCD enzyme is not required for spacer preparation and that RecA inhibits adaptation probably because it prevents DNA processing and stimulates homologous recombination. However, helicase activity of RecBC(D) is required and is helped by 3’-5’ host ssDNA exonucleases to occasionally generate appropriate DNA substrates for Cas1-Cas2 binding. Our in vitro analysis implies that Cas1-Cas2 forms a stable complex on DNA substrates with 5’ overhangs and catalyses their cutting. Overall, our data suggest that 5’ overhangs are important as substrates for adaptation and that these may be bound and processed by Cas1-Cas2.

CRISPR-Cas ; RecBCD ; Cas1-2 ; E. coli ; nucleases

nije evidentirano