Cyclic nucleotide signalling is a key component of anti-viral defence in all domains of life, from bacteria to humans. Viral detection activates a nucleotide cyclase to generate a second messenger, resulting in activation of effector proteins. This is exemplified by the metazoan cGAS-STING innate immunity pathway 1 , which originated in bacteria 2 . These defence systems require a sensor domain such as STING or SAVED to bind the cyclic nucleotide, coupled with an effector domain that causes cell death when activated by destroying essential biomolecules 3 . One example is the TIR (Toll/interleukin-1 receptor) domain, which degrades the essential cofactor NAD + when activated in response to pathogen invasion in plants and bacteria 2,4,5 or during nerve cell programmed death 6 . Here, we show that a bacterial anti-viral defence system generates a cyclic tri-adenylate (cA 3 ) signal which binds to a TIR-SAVED effector, acting as the "glue" to allow assembly of an extended superhelical solenoid structure. Adjacent TIR subunits interact to organise and complete a composite active site, allowing NAD + degradation. Our study illuminates a striking example of largescale molecular assembly controlled by cyclic nucleotides and reveals key details of the mechanism of TIR enzyme activation.Recent discoveries have revealed a central role for cyclic nucleotide second messengers in prokaryotic anti-viral defence by type III CRISPR 7,8 , CBASS (Cyclic nucleotide Based Antiphage Signalling Systems) 3 and PYCSAR (Pyrimidine Cyclase System for Antiphage Resistance) 9 . These systems activate potent effector proteins that destroy key cellular components such as nucleic acids, cofactors or membranes to disrupt viral replication 3,10 . A good example is the TIR domain, which functions as an enzyme that degrades NAD + to cause cell death in plants infected with pathogens 5,11 , anti-phage immunity in the bacterial Thoeris 12,13 and TIR-STING 2 systems, and programmed nerve cell death in metazoa 6 . TIR domain activation requires effector subunit assembly, but the molecular mechanisms are still not fully understood..
In Archaea, the proteins involved in the genetic information processing pathways, including DNA replication, transcription, and translation, share strong similarities with those of eukaryotes. Characterizations of components of the eukaryotic-type replication machinery complex provided many interesting insights into DNA replication in both domains. In contrast, DNA repair processes of hyperthermophilic archaea are less well understood and very little is known about the intertwining between DNA synthesis, repair and recombination pathways. The development of genetic system in hyperthermophilic archaea is still at a modest stage hampering the use of complementary approaches of reverse genetics and biochemistry to elucidate the function of new candidate DNA repair gene. To gain insights into genomic maintenance processes in hyperthermophilic archaea, a protein-interaction network centred on informational processes of Pyrococcus abyssi was generated by affinity purification coupled with mass spectrometry. The network consists of 132 interactions linking 87 proteins. These interactions give insights into the connections of DNA replication with recombination and repair, leading to the discovery of new archaeal components and of associations between eucaryotic homologs. Although this approach did not allow us to clearly delineate new DNA pathways, it provided numerous clues towards the function of new molecular complexes with the potential to better understand genomic maintenance processes in hyperthermophilic archaea. Among others, we found new potential partners of the replication clamp and demonstrated that the single strand DNA binding protein, Replication Protein A, enhances the transcription rate, in vitro, of RNA polymerase. This interaction map provides a valuable tool to explore new aspects of genome integrity in Archaea and also potentially in Eucaryotes.
Several archaeal species prevalent in extreme environments are particularly exposed to factors likely to cause DNA damages. These include hyperthermophilic archaea (HA), living at temperatures >70°C, which arguably have efficient strategies and robust genome guardians to repair DNA damage threatening their genome integrity. In contrast to Eukarya and other archaea, homologous recombination appears to be a vital pathway in HA, and the Mre11–Rad50 complex exerts a broad influence on the initiation of this DNA damage response process. In a previous study, we identified a physical association between the Proliferating Cell Nuclear Antigen (PCNA) and the Mre11–Rad50 (MR) complex. Here, by performing co-immunoprecipitation and SPR analyses, we identified a short motif in the C- terminal portion of Pyrococcus furiosus Mre11 involved in the interaction with PCNA. Through this work, we revealed a PCNA-interaction motif corresponding to a variation on the PIP motif theme which is conserved among Mre11 sequences of Thermococcale species. Additionally, we demonstrated functional interplay in vitro between P. furiosus PCNA and MR enzymatic functions in the DNA end resection process. At physiological ionic strength, PCNA stimulates MR nuclease activities for DNA end resection and promotes an endonucleolytic incision proximal to the 5′ strand of double strand DNA break.
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