engleski

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

The CRISPR-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a crRNA-mediated interference mechanism. In Type I CRISPR-Cas systems crRNA guided by a Cascade complex recognises the matching target DNA and promotes an R-loop formation, RNA-DNA hybrid. The helicase-nuclease Cas3 protein is then recruited to the Cascade/R-loop complex where it nicks and degrades DNA. R-loop is also an intermediate in replication of ColE1 type of plasmids and Cas3 has been shown to induce uncontrolled runaway replication when overexpressed from them. Cas3 ATPase/helicase activity is required in this process while other components of the CRISPR-Cas immunity are not. Since Cas3 has insufficient activity in CRISPR-Cas immunity at 37°C, we asked if the temperature of incubation will influence Cas3-induced uncontrolled plasmid replication. Plasmid replication was studied by comparing the plasmid yields in cells known to have temperature dependent CRISPR-Cas immunity grown at 30 °C and 37 °C (wt, hns, htpG, hns htpG). We found that Cas3-induced uncontrolled plasmid replication is temperature dependent and occurs only at 37 °C independently of the cell’s genotype. It is dependent on the Cas3 helicase activity which seems to be inhibited at 30 °C. We also found that the plasmid replication at 30 °C is strongly reduced by the hns mutation.

CRISPR-Cas, Cas3, ColE1 plasmids, temperature

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