Molecular Mechanisms Of The Nucleic Acid Interference By The Sulfolobus Type ?-B Cmr-? Complex

CRISPR-Cas(clustered regularly interspaced short palindromic repeats-CRISPR-associated)systems constitute a unique antiviral immunity in bacteria and archaea.The systems are able to protect their prokaryotic hosts from the invasion of foreign genetic elements,such as plasmids and viruses.Each CRISPR-Cas system consists of at least two genetic units: the CRISPR clusters and the cas genes.In the latest classification,CRISPR-Cas systems are classified into two general classes and at least six different main types.Of these,type ? CRISPR-Cas systems show both RNA cleavage and DNA cleavage activity,representing the only known CRISPR-Cas system with the dual nucleic acid interference activity.Previous studies have shown that the ?-B Cmr-? system of Sulfolobus islandicus is able to cleave the target RNA at regular intervals of 6-nt nucleotides,and upon the activation by the binding of cognate target RNA,the tertiary complex(Cmr-?::target RNA)show a high unspecific ssDNA cleavage activity.However,little is known about how the dual nucleic acid(NA)cleavage by Cmr-? system is regulated.In the thesis,it was hypothesized that Cmr1? could function as an integral activation module to enhance the NA interference by Cmr-? system,and that Cmr4? could play an essential role in the spatiotemporal regulation of the complex activation in the Cmr-? immune process.To investigate the function of Cmr1?,a nonessential subunit of Cmr complex,a deletion mutant ???1? was constructed with the genetic host of S.islandicus REY15 A.In vivo interference activity analysis revealed that ???1? mutant was still active in the target RNA interference activity and invader plasmid silencing activity albeit at a much reduced level.A stable nucleoprotein complex Cmr-??1(Cmr2?-6?),which does not consist of any Cmr1? protein,was purified.In vitro biochemical analysis revealed that the crRNA in the Cmr-??1 complex was degenerated,but the complex still had weak activity of ssRNA and ssDNA,consistent with the in vivo data of the deletion mutant.Comparison of amino acid sequence of Cmr1 proteins led to the identification of several conserved amino acid residues in the first ?-helix of the N-terminus of Cmr1?,which are located in the surface groove of Cmr1? by protein structure modeling.Several of these conserved amino acids were subjected to substitution mutagenesis.The resulting cmr1? mutants were analyzed for invader plasmid assay whereas mutated RNPs were purified from the mutants and tested for NA cleavage in vitro.The results showed that the two conserved hydrophobic amino acids(W58 and F59)of Cmr1? have an important function in mediating DNA interference in vivo and ssDNA cleavage in vitro,probably by affecting the binding of Cmr1? to the target RNA.The other four highly conserved amino acid residues(I52,G54,R57,and R61)also showed an influence on the interference activity in vivo and the cleavage activity of the target RNA in vitro,and they probably function in the binding of Cmr1? to crRNA.Although Cmr1? is not essential for the Cmr-? complex,upon binding to the core Cmr-? complex,the protein greatly enhances the antiviral interference activity by binding the crRNA with the target RNA.In addition,the correlation between the appearance of Cmr1 and the thermophilic lifestyle of microbial hosts may also reflect the co-evolution of the type ?-B CRISPR-Cas system and the thermophilic life.The Cas10 family proteins(Cmr2 and Csm1)are the type-specific protein of type ? CRISPR-Cas systems,in which two conserved domains are responsible for the non-specific ssDNA cleavage and second messenger cOAs synthesis that activate non-specific ssRNA cleavage by Csx1,a CARF domain nuclease.Our experimental data indicated that the Palm2 domain of Cmr2? plays a fundamental role in silencing the invasion of foreign genetic elements,which exhibits a low expression.However,when the invader is expressed actively,the HD domain of Cmr2? is significantly activated to kill the host cell.Cmr4 is the large subunit of the backbone proteins in ?-B Cmr complexes and is essential to the assembly and stability of the effector complex.A number of conserved amino acid residues were identified by multiple sequence alignment,and substitution mutation analysis of these Cmr4? conserved amino acids strongly impaired the NA interference activity of the Cmr-? system in vivo.Purification of the effector complex from each mutant revealed that a stable Cmr-? complex was only purified from two cmr4a mutants(Cmr4aH16A,Cmr4aD83A),suggesting most of the cmr4a mutations could have impaired the structure of the resulting mutated effector complexes.Furthermore,we show that small Cmr-a complexes only containing three Cmr4? could be the predominant species of the effector complexes,which is in contrast to the wild-type Cmr-? complex in which the large Cmr-a complex having 4 Cmr4a subunits dominates.Nevertheless,in vitro NA cleavage pattern by the mutated RNP was very similar to that of the wild-type RNP.To yield a further insight into synthetic lethality induced by the Cmr4aD27A substitution mutation,we attempted to construct the D27 A substitution in the ??4?-D83 A strain,and a double mutant named ??4?-D27/83 A was obtained.However,consistent efforts to purify a Cmr-a complex from the double mutant failed.These results suggest that the D83 A substitution may affect the stability of the Cmr-? complex,or the D27 A substitution can further exacerbate the instability of the Cmr-a complex.Furthermore,the D27 A substitution was also successful in the double mutant of the HD and Palm2 domains of Cmr2a,which is inactive in both the HD nuclease and the c-A4 synthesis.Gel retardation assay showed the nuclease-dead mutant showed a distinctive pattern of Cmr-a::target RNA complexes,yielding important insights into the target RNA binding,cleavage and product release.Type ? CRISPR-Cas system is recognized as an immune system that relies on target RNA activation of unspecific ssDNA cleavage and inactivation of the robust DNase upon target RNA cleavage and release of products.To avoid self-immunization,type ? nucleoprotein complexes cleave the target RNA and ssDNA at a very rapid rate.We reason that the Cmr-? complex with Cmr4?D27A substitution,which cannot efficiently cleave the cognate target RNA,degrades non-specific ssDNA continuously,leading to an irreversibly damage to the genome and subsequently death to the host cell.Taken together,these results indicate that Cmr4? plays a crucial role in the spatiotemporal regulation of the Cmr-a nuclease and cyclase activities during the Cmr-? immune process.