Activity And Function Of DHH-DHHA1 Family Nuclease In Sulfolobus Islandicus

CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats and CRISPR-associated genes)systems are adaptive immune systems encoded by bacteria and archaea.In this study,we employ the Sulfolobus islandicus as the material,which encodes type III CRISPR-Cas system.When foreign nucleic acids invading,type III CRISPR-Cas system is activated for target RNA cleavage,ss DNA cleavage and synthesis of cyclic oligo-adenylate(cOA)activity to perform immune response.Among them,cOA is a second massenger that specifically activates the non-specific RNase activity in vivo to inhibit virus proliferation.However,the RNase activity activated by cOA is non-specific which could induce cell dormancy or even death.So cOA should be cleared on time after the immune response.It has been found that a protein called ring nuclease containing a special CARF domain,is encoded by the Sulfolobus solfataricus.This protein can specifically bind to cOA and degrade it.In our previous research,we found that Sulfolobus islandicus possesses cOA degrading enzyme activity that is different from ring nuclease and dependent on metal ions.On the basis of the previous research,we conducted the investigation of the cOA degrading enzyme in Sulfolobus islandicus,and further explored the molecular mechanisms and its functions in vivo.The experimental results contain:1.The fractions from previous experiments were separated and purified step by step and identified by mass spectrometry,and 4 candidate proteins that might be cOA degrading enzymes were found.The four proteins were expressed and purified separately,and investigated their activity.Finally,it was found that only SiRe?0244 exhibited cOA degradation activity.After further research,we confirmed that SiRe?0244 is a cOA degrading enzyme that is related to the cell membrane and belongs to the DHH-DHHA1 family,named MAD(a membrane-associated DHH-DHHA1 family nuclease).MAD degrades cOA at a high level,while ring nuclease efficiently degrades low-level cOA.2.The prominent members of DHH-DHHA1 family include single-strand specific exonuclease,nano RNases,cell cycle protein etc.They play an important role in various cellular processes like nucleic acid metabolism,DNA recombination,DNA repair,homeostasis maintenance and stress tolerance.In our research,we found that SiRe?0244has nuclease activities that substrates contain ss RNA,ds RNA,ss DNA,ds DNA.And among the three conserved motifs of SiRe?0244 protein,Mutation of the motifs yielded three SiRe?0244 mutants.M1,D10A-D12A;M2,D87A-H88A-H89A;M3,H271 A.The D10A-D12 A mutant protein loses degradation activities of ss DNA,and ds DNA.The D87A-H88A-H89 A mutant protein loses all nucleic acid degradation activities.While the H271 A mutant protein loses degradation activities of ss DNA and ds DNA.3.Further,we used ??E233 strain as a genetic host to construct SiRe?0244 deletion strain.We performed transcriptome sequencing of the wild-type strain and mutant strain and found that SiRe?0244 can indirectly regulate the expression of flagella-related genes.The results were confirmed by q RT-PCR.The phenotypes of wild-type strains and mutant revealed that the absence of SiRe?0244 could promote the formation of flagella of the strain and the formation of other unknown substances on the cell membrane surface.In summary,this study detected and identified MAD protein in Sulfolobus islandicus.This enzyme has high cOA degrades activity and may cooperate with ring nuclease to participate in the immune regulation of the CRISPR-Cas system.Moreover,this enzyme is the member of DHH-DHHA1 family and has high nuclease activity.Therefore,in addition to degrading cOA,this enzyme may also participate in other functions in vivo.This study also proved that SiRe?0244 inhibits the expression and of flagella-related genes in Sulfolobus islandicus and the formation of other unknown subatances on the surface,but the specific mechanism of regulation is not yet clear.