Cas3 stimulates runaway replication of a ColE1 plasmid inEscherichia coliand antagonises RNaseHI

Abstract

Cas3 nuclease-helicase is part of CRISPR immunity systems in many bacteria and archaea. In type I CRISPR, Cas3 nuclease degrades invader DNA that has been base-paired to crRNA as an R-loop within a "Cascade" complex. An R-loop is a DNA-RNA hybrid that includes a displaced single-strand DNA loop. Purified Cas3 from E. coli and the archaeon M. thermautrophicus can process R-loops without DNA/RNA sequence specificity and without Cascade. This has potential to affect other aspects of microbial biology that involve R-loops. Regulatory RNAs and host cell proteins modulate replication of ColE1 plasmids (e.g., pUC) from R-loop primers. We observed that Cas3 could override endogenous control of a ColE1 replicon, stimulating uncontrolled ("runaway") replication and resulting in much higher plasmid yields. This effect was absent when using helicase-defective Cas3 (Cas3 (K320L) ) or a non-ColE1 plasmid, and was dependent on RNaseHI. Cas3 also promoted formation of plasmid multimers or concatemers, a phenotype consistent with deregulated ColE1 replication and typical of cells lacking RNaseHI. These effects of Cas3 on ColE1 plasmids are inconsistent with it unwinding R-loops in vivo, at least in this assay. We discuss a model of how Cas3 might be able to regulate RNA molecules in vivo, unless it is targeted to CRISPR defense by Cascade, or kept in check by RecG and RNaseHI.

NilD CRISPR RNA contributes to Xenorhabdus nematophila colonization of symbiotic host nematodes

“…In other systems cas1 and cas2 genes are not necessary for CRISPR RNA processing or activity, but rather encode nucleases that form a complex necessary for acquisition of new spacers (Fineran and Charpentier, 2012;Nuñez et al, 2014). In addition, cas1 is involved in DNA repair and chromosome segregation (Babu et al, 2010), while cas3 promotes plasmid replication in E. coli (Ivancic-Bace et al, 2013).…”

Cas3 Protein—A Review of a Multi-Tasking Machine

“…An interesting set of observations has been made implicating Cas3 in several other cellular events that center on, or are at least associated with, regulatory processing of RNA molecules. In one example, ectopic over-expression of Cas3 in E. coli cells stimulated uncontrolled DNA replication of plasmids from their ColE1 replicons, but had no such effect on non-ColE1 plasmids [76]. These plasmids use R-loop at ori to initiate replication formed by plasmid encoded RNA molecules, one (Δhns) that are engineered to target λ phage are effective at resisting infection at 30 °C but not 37 °C (Figure 3), but cells lacking HtpG and HNS are phage-sensitive.…”

Interplay between base excision repair activity and toxicity of 3-methyladenine DNA glycosylases in an E. coli complementation system

“…Formation of concatemers was identified in the form of high molecular weight bands on the gel that disappear upon restriction as previously described in [22]. …”