| Abstract [eng] |
Type III CRISPR-Cas systems employ Csm or Cmr ribonucleoprotein complexes to cleave foreign RNA and DNA target to confer immunity against viruses and other nucleic acids. By recognizing target RNA Csm/Cmr also synthesizes 3-6 nt cyclic oligoadenylates (cOA) from ATP. Either cA4 or cA6 allosterically activates CARF domain-containing accessory proteins. cOA ligand binding to a CARF sensor results in activation of various effector domains that can possess non-specific ribonuclease, deoxyribonucklease, deaminase and other enzymatic activities. These ancillary effectors together with Csm/Cmr provide immunity to its host, however they can also be toxic to a cell and result in altruistic suicide unless extant cOA’s are removed after infection is over. Recently characterized enzymes called ring nucleases degrade cA4 or cA6. At this point several families of ring nucleases that differ in protein fold, reaction mechanism and efficiency are characterized. Some uncharacterized CRISPR-associated genes are also hypothesized to possess ring nuclease activity. The aim of this work was to examine an activity of hypothetical ring nuclease Csx20 in vitro and in vivo. We discovered that similarly to other ring nucleases bacterial Csx20 exists in a dimeric state in solution. Although present as a dimer, Csx20 homologue from a virus is found also in trimeric, tetrameric, and hexameric states. We showed that Csx20 catalyze metal-independent hydrolysis of cA4 producing cAMP. We also determined that Csx20 are efficient enzymes compared to Crn1 ring nucleases. By changing hypothetical active site residue His9 to alanine the observed activity was significantly reduced. Lastly, we demonstrate that both of our tested Csx20 abolish growth restriction of E. coli caused by type III CRISPR cA4-effector. Together these results show that proteins of Csx20 family are ring nucleases. |
