Department of Biology, Faculty of Science, University of Zagreb, Horvatovac 102a, 10000 Zagreb, Croatia.
FEMS Microbiol Lett. 2019 May 1;366(9). doi: 10.1093/femsle/fnz106.
The clustered regularly interspersed short palindromic repeats (CRISPR)-Cas system constitutes an adaptive immunity system of prokaryotes against mobile genetic elements using a CRISPR RNA (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. The Cas3 activity in CRISPR-Cas immunity is reduced in Δhns cells at 37°C for unknown reasons. Cas3 can also influence regulation of plasmid replication and promote uncontrolled ('runaway') replication of ColE1 plasmids independently of other CRISPR-Cas components, requiring only its helicase activity. In this work we wanted to test whether Cas3-stimulated uncontrolled plasmid replication is affected by the temperature in Δhns and/or ΔhtpG mutants. We found that Cas3-stimulated uncontrolled plasmid replication occurs only at 37°C, irrespective of the genotype of the analysed mutants, and dependent on Cas3 helicase function. We also found that plasmid replication was strongly reduced by the hns mutation at 30°C and that Cas3 could interfere with T4 phage replication at both incubation temperatures.
成簇规律间隔短回文重复序列 (CRISPR)-Cas 系统构成了原核生物针对移动遗传元件的适应性免疫系统，利用 CRISPR RNA (crRNA) 介导的干扰机制。在 I 型 CRISPR-Cas 系统中，由级联复合物引导的 crRNA 识别匹配的靶 DNA 并促进 R 环形成，即 RNA-DNA 杂交。然后，解旋酶-核酸酶 Cas3 蛋白被招募到级联/R 环复合物中，在那里它切割并降解 DNA。由于未知原因，在 37°C 下，Δhns 细胞中的 CRISPR-Cas 免疫中的 Cas3 活性降低。Cas3 还可以影响质粒复制的调节，并独立于其他 CRISPR-Cas 成分促进 ColE1 质粒的失控（“失控”）复制，仅需要其解旋酶活性。在这项工作中，我们想测试 Cas3 刺激的失控质粒复制是否受 Δhns 和/或 ΔhtpG 突变体中温度的影响。我们发现，Cas3 刺激的失控质粒复制仅在 37°C 时发生，与分析突变体的基因型无关，并且依赖于 Cas3 解旋酶功能。我们还发现，在 30°C 时 hns 突变强烈降低了质粒复制，并且 Cas3 可以在两个孵育温度下干扰 T4 噬菌体复制。
