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Cas3-stimulated runaway replication of modified ColE1 plasmids in Escherichia coli is temperature dependent

The clustered regularly interspersed short palindromic repeats (CRISPR) and nearby CRISPR- associated (Cas) genes provide bacteria and archea with an adaptive immunity system against foreign genetic elements (plasmids and phages). The immunity is based on integration of a small piece of foreign DNA (spacer) within the CRISPR locus which is then transcribed and processed into CRISPR RNA (crRNA) which, guided by Cas proteins, base-pairs to foreign DNA resulting in its degradation. In type-I CRISPR-Cas systems of Escherichia coli, crRNA guided by the Cascade complex recognises the complementary target DNA and promotes an R-loop formation, RNA-DNA hybrid. Upon R-loop formation, the helicase/nuclease Cas3 protein is recruited which then nicks and degrades foreign DNA. This Cas3 activity in CRISPR-Cas immunity was found to be effective in Δhns cells at 30°C, but reduced at 37°C for unknown reasons. Besides its role in CRISPR-Cas immunity, Cas3 protein can also provoke uncontrolled ("runaway") replication of ColE1 type plasmids, whose initiation of replication includes R-loop formation, in a manner independent of other CRISPR-Cas components. The aim of this work was to test whether the effect of Cas3 protein on the replication of ColE1 type plasmids was also temperature dependent in Δhns and/or ΔhtpG mutants. Our results show that Cas3- stimulated replication of ColE1 plasmids occurs only at 37°C and is independent of the genotype of the analysed mutants, but is dependent on the Cas3 helicase function. In addition, we found that plasmid replication is reduced in Δhns mutants at 30°C.

CRISPR, Cas3, ColE1, replication, E. coli

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