RESUMO
The repair of DNA double-strand breaks (DSBs) is essential to maintaining the integrity of the genome, and organisms have evolved a conserved mechanism to facilitate their repair. In eukaryotes, archaea, and some bacteriophage, a complex made up of Mre11 and Rad50 (MR complex), which are a nuclease and ATPase, respectively, is involved in the initial processing of DSBs. Rad50 is a member of the ATP Binding Cassette (ABC) protein superfamily, the members of which contain an important Signature motif that acts in trans to complete the dimeric ATP binding site. To explore the functional relevance of this motif, four of its five residues were mutated in bacteriophage T4 Rad50, and their respective ATPase and nuclease activities were evaluated. The mutations reveal the functional roles of the Signature motif in ATP binding, hydrolysis, and cooperativity. In several mutants, the degree of DNA activation of ATP hydrolysis activity is reduced, indicating that the Signature motif is involved in allosteric signal transmission between the DNA and ATP binding sites of the MR complex. ATP hydrolysis is not required for nuclease activity when the probe is near the beginning of the DNA substrate; however, when an internal probe is used, decreases in ATPase activity have substantial effects on nuclease activity, suggesting that ATP hydrolysis is involved in translocation of the complex. Unexpectedly, the ATP hydrolysis and nuclease activities are not directly correlated with each other, and each mutation appears to differentially affect the exonuclease activity of Mre11.
Assuntos
Adenosina Trifosfatases/química , Bacteriófago T4/enzimologia , Complexos Multienzimáticos/química , Pyrococcus furiosus/virologia , Proteínas Virais/química , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/química , Trifosfato de Adenosina/genética , Regulação Alostérica/genética , Motivos de Aminoácidos/genética , Bacteriófago T4/genética , Cristalografia por Raios X , Reparo do DNA/genética , Exodesoxirribonucleases/química , Exodesoxirribonucleases/genética , Complexos Multienzimáticos/genética , Mutagênese Sítio-Dirigida , Transdução de Sinais/genética , Proteínas Virais/genéticaRESUMO
The small CRISPR-derived RNAs of bacteria and archaea provide adaptive immunity by targeting the DNA of invading viruses and plasmids. Hale et al. (2009) now report on a new variant CRISPR/Cas complex in the archaeon Pyrococcus furiosus that uses guide RNAs to specifically target and cleave RNA not DNA.
Assuntos
Pyrococcus furiosus/genética , Pyrococcus furiosus/imunologia , Interferência de RNA , RNA Arqueal/imunologia , Pyrococcus furiosus/virologia , RNA Arqueal/genética , RNA Viral/imunologia , Pequeno RNA não TraduzidoRESUMO
Compelling evidence indicates that the CRISPR-Cas system protects prokaryotes from viruses and other potential genome invaders. This adaptive prokaryotic immune system arises from the clustered regularly interspaced short palindromic repeats (CRISPRs) found in prokaryotic genomes, which harbor short invader-derived sequences, and the CRISPR-associated (Cas) protein-coding genes. Here, we have identified a CRISPR-Cas effector complex that is comprised of small invader-targeting RNAs from the CRISPR loci (termed prokaryotic silencing (psi)RNAs) and the RAMP module (or Cmr) Cas proteins. The psiRNA-Cmr protein complexes cleave complementary target RNAs at a fixed distance from the 3' end of the integral psiRNAs. In Pyrococcus furiosus, psiRNAs occur in two size forms that share a common 5' sequence tag but have distinct 3' ends that direct cleavage of a given target RNA at two distinct sites. Our results indicate that prokaryotes possess a unique RNA silencing system that functions by homology-dependent cleavage of invader RNAs.
Assuntos
Proteínas Arqueais/imunologia , Pyrococcus furiosus/imunologia , Interferência de RNA , RNA Arqueal/imunologia , Proteínas Arqueais/metabolismo , Sequência de Bases , Pyrococcus furiosus/genética , Pyrococcus furiosus/metabolismo , Pyrococcus furiosus/virologia , RNA Arqueal/química , RNA Arqueal/genética , RNA Arqueal/metabolismo , RNA Viral/imunologia , Pequeno RNA não TraduzidoRESUMO
Pyrococcus furiosus is a hyperthermophilic archaeal microorganism found near deep-sea thermal vents and its optimal growth temperature of 100 degrees C. Recently, a 38.8-kDa protein from P. furiosus DSM 3638 was isolated and characterized. Electron microscopy revealed that this protein aggregated as spheres of approximately 30 nm in diameter, which we designated P. furiosus virus-like particles (PfVs). X-ray crystallographic analysis at 3.6-A resolution revealed that each PfV consisted of 180 copies of the 38.8-kDa protein and retained T=3 icosahedral symmetry, as is often the case in spherical viruses. The total molecular mass of each particle was approximately 7 MDa. An examination of capsid structures suggested strong evolutionary links among PfV, tailed double-stranded DNA bacteriophages, and herpes viruses. The similar three-dimensional structures of the various coat proteins indicate that these viral capsids might have originated and evolved from a common ancestor. The structure of PfV provides a previously undescribed example of viral relationships across the three domains of life (Eukarya, Bacteria, and Archaea).
