title: Cas3 stimulates runaway replication of a ColE1 plasmid in Escherichia coli and 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.
authors: Faculty of Science, Department of Molecular Biology, University of Zagreb, Zagreb, Croatia. ivana.ivancic.bace@biol.pmf.hr
authors: Ivana
authors: I
authors: Ivančić-Baće
authors: Marin
authors: M
authors: Radovčić
authors: Luka
authors: L
authors: Bočkor
authors: Jamieson L
authors: JL
authors: Howard
authors: Edward L
authors: EL
authors: Bolt
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D064130:CRISPR-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D064113:CRISPR-Cas Systems; D004265:DNA Helicases / Q000235:genetics / Q000378:metabolism*; D004261:DNA Replication; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D048168:Escherichia coli K12 / Q000235:genetics* / Q000378:metabolism; D029968:Escherichia coli Proteins / Q000235:genetics / Q000378:metabolism*; D016254:Mutagenesis, Insertional; D009690:Nucleic Acid Conformation; D010957:Plasmids / Q000737:chemistry / Q000235:genetics*; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012093:Replicon; D016914:Ribonuclease H / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D064130:CRISPR-Associated Proteins; D004269:DNA, Bacterial; D029968:Escherichia coli Proteins; D012329:RNA, Bacterial; C042833:ROM protein, Bacteria; C071446:RecG protein, E coli; D016914:Ribonuclease H; C078856:ribonuclease HI; D004265:DNA Helicases; C574631:ygcB protein, E coli
keywords: CRISPR; Cas3; ColE1; R-loop; RNaseHI; RecG