Sulfolobus islandicus codes for four DNA polymerases: three are of the B-family (Dpo1, Dpo2, and Dpo3), and one is of the Y-family (Dpo4). Western analysis revealed that among the four polymerases, only Dpo2 exhibited DNA damage-inducible expression. To investigate how these DNA polymerases could contribute to DNA damage tolerance in S. islandicus, we conducted genetic analysis of their encoding genes in this archaeon. Plasmid-borne gene expression revealed that Dpo2 increases cell survival upon DNA damage at the expense of mutagenesis. Gene deletion studies showed although dpo1 is essential, the remaining three genes are dispensable. Furthermore, although Dpo4 functions in housekeeping translesion DNA synthesis (TLS), Dpo2, a B-family DNA polymerase once predicted to be inactive, functions as a damage-inducible TLS enzyme solely responsible for targeted mutagenesis, facilitating GC to AT/TA conversions in the process. Together, our data indicate that Dpo2 is the main DNA polymerase responsible for DNA damage tolerance and is the primary source of targeted mutagenesis. Given that crenarchaea encoding a Dpo2 also have a low-GC composition genome, the Dpo2-dependent DNA repair pathway may be conserved in this archaeal lineage.

Type III CRISPR-Cas systems show the target (tg)RNA-activated indiscriminate DNA cleavage and synthesis of oligoadenylates (cOA) and a secondary signal that activates downstream nuclease effectors to exert indiscriminate RNA/DNA cleavage, and both activities are regulated in a spatiotemporal fashion. In III-B Cmr systems, cognate tgRNAs activate the two Cmr2-based activities, which are then inactivated via tgRNA cleavage by Cmr4, but how Cmr4 nuclease regulates the Cmr immunization remains to be experimentally characterized. Here, we conducted mutagenesis of Cmr4 conserved amino acids in Saccharolobus islandicus, and this revealed that Cmr4α RNase-dead (dCmr4α) mutation yields cell dormancy/death. We also found that plasmid-borne expression of dCmr4α in the wild-type strain strongly reduced plasmid transformation efficiency, and deletion of CRISPR arrays in the host genome reversed the dCmr4α inhibition. Expression of dCmr4α also strongly inhibited plasmid transformation with Cmr2αHD and Cmr2αPalm mutants, but the inhibition was diminished in Cmr2αHD,Palm. Since dCmr4α-containing effectors lack spatiotemporal regulation, this allows an everlasting interaction between crRNA and cellular RNAs to occur. As a result, some cellular RNAs, which are not effective in mediating immunity due to the presence of spatiotemporal regulation, trigger autoimmunity of the Cmr-α system in the S. islandicus cells expressing dCmr4α. Together, these results pinpoint the crucial importance of tgRNA cleavage in autoimmunity avoidance and in the regulation of immunization of type III systems.

Highlights d Metal-dependent cOA degradation activity accelerates clearance of high-level cOA d The metal-dependent activity is associated with the cell membrane d A membrane-associated DHH-DHHA1 family nuclease (MAD) is found to degrade cOA d cOA degradation of MAD deactivates the type III CRISPR accessory nuclease

Cas12a is a type V-A CRISPR-Cas RNA-guided endonuclease. It cleaves dsDNA at specific site, and then is activated for nonspecific ssDNA cleavage in trans in vitro. The immune function of the trans activity is still unknown. To address this question, we constructed a Cas12a targeting system in Escherichia coli, where Cas12a cleaved a high-copy target plasmid to unleash the trans ssDNA cleavage activity. Then, we analyzed the effect of the Cas12a targeting on a non-target plasmid and a ssDNA phage. The results show that Cas12a efficiently eliminates target plasmid but exerts no impact on the maintenance of the non-target plasmid or plague formation efficiency of the phage. In addition, a two-spacer CRISPR array, which facilitates target plasmid depletion, still has no detectable effect on the non-target plasmid or phage either. Together, the data suggest that the trans ssDNA cleavage of Cas12a does not contribute to immunity in vivo.

Summary Type III CRISPR-cas systems initiate cyclic oligo-adenylate (cOA) signaling to initiate immune response. Previously, we identified that a membrane-associated DHH-DHHA1 family protein from Sulfolobus islandicus efficiently degrades cOA. Here, we provide detailed protocols for expression and purification of the protein from its native host and a cOA degradation assay with the purified enzyme. The methodology should be of interest for researchers studying Sulfolobus , membrane-associated proteins, or type III CRISPR-cas systems. For complete details on the use and execution of this protocol, please refer to Zhao et al. (2020) .