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CRISPR-Cas systems confer adaptive immunity in prokaryotes, facilitating the recognition and destruction of invasive nucleic acids. Type III CRISPR systems comprise large, multisubunit ribonucleoprotein complexes with a catalytic Cas10 subunit. When activated by the detection of foreign RNA, Cas10 generates nucleotide signalling molecules that elicit an immune response by activating ancillary effector proteins. Among these systems, the Bacteroides fragilis type III CRISPR system was recently shown to produce a novel signal molecule, SAM-AMP, by conjugating ATP and SAM. SAM-AMP regulates a membrane effector of the CorA family to provide immunity. Here, we focus on NYN, a ribonuclease encoded within this system, probing its potential involvement in crRNA maturation. Structural modelling and in vitro ribonuclease assays reveal that NYN displays robust sequence-nonspecific, Mn2+-dependent ssRNA-cleavage activity. Our findings suggest a role for NYN in trimming crRNA intermediates into mature crRNAs, which is necessary for type III CRISPR antiviral defence. This study sheds light on the functional relevance of CRISPR-associated NYN proteins and highlights the complexity of CRISPR-mediated defence strategies in bacteria. [ABSTRACT FROM AUTHOR]

There has long been a fascination in the DNA repair pathways of archaea, for two main reasons. Firstly, many archaea inhabit extreme environments where the rate of physical damage to DNA is accelerated. These archaea might reasonably be expected to have particularly robust or novel DNA repair pathways to cope with this. Secondly, the archaea have long been understood to be a lineage distinct from the bacteria, and to share a close relationship with the eukarya, particularly in their information processing systems. Recent discoveries suggest the eukarya arose from within the archaeal domain, and in particular from lineages related to the TACK superphylum and Lokiarchaea. Thus, archaeal DNA repair proteins and pathways can represent a useful model system. This review focuses on recent advances in our understanding of archaeal DNA repair processes including base excision repair, nucleotide excision repair, mismatch repair and double-strand break repair. These advances are discussed in the context of the emerging picture of the evolution and relationship of the three domains of life. [ABSTRACT FROM AUTHOR]

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR-Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also discuss the differences in terms of mechanisms between the subtype III-B systems in S. solfataricus and Pyrococcus furiosus. [ABSTRACT FROM AUTHOR]

Several unexpected reports of iron–sulphur clusters in nucleic acid binding proteins have recently appeared in the literature. Once thought to be relatively rare in these systems, iron–sulphur clusters are now known to be essential components of diverse nucleic acid processing machinery including glycosylases, primases, helicases, nucleases, transcription factors, RNA polymerases and RNA methyltransferases. In many cases, the function of the cluster is poorly understood and crystal structures of these iron–sulphur enzymes reveal little in common between them. In this article, we review the recent developments in the field and discuss to what extent there might exist common mechanistic roles for iron–sulphur clusters in nucleic acid enzymes. [Copyright &y& Elsevier]

Abstract: Nucleotide excision repair (NER) pathways remove a wide variety of bulky and helix-distorting lesions from DNA, and involve the coordinated action of damage detection, helicase and nuclease proteins. Most archaeal genomes encode eucaryal-type NER proteins, including the helicases XPB and XPD and nuclease XPF. These have been a valuable resource, yielding important mechanistic and structural insights relevant to human health. However, the nature of archaeal NER remains very uncertain. Here we review recent studies of archaeal NER proteins relevant to both eucaryal and archaeal NER systems and the evolution of repair pathways. [ABSTRACT FROM AUTHOR]

XPB helicase is an integral part of transcription factor TFIIH, required for both transcription initiation and nucleotide excision repair (NER). Along with the XPD helicase, XPB plays a crucial but only partly understood role in defining and extending the DNA repair bubble around lesions in NER. Archaea encode clear homologues of XPB and XPD, and structural studies of these proteins have yielded key insights relevant to the eukaryal system. Here we show that archaeal XPB functions with a structure-specific nuclease, Bax1, as a helicase-nuclease machine that unwinds and cleaves model NER substrates. DNA bubbles are extended by XPB and cleaved by Bax1 at a position equivalent to that cut by the XPG nuclease in eukaryal NER. The helicase activity of archaeal XPB is dependent on the conserved Thumb domain, which may act as the helix breaker. The N-terminal damage recognition domain of XPB is shown to be crucial for XPB-Bax1 activity and may be unique to the archaea. These findings have implications for the role of XPB in both archaeal and eukaryal NER and for the evolution of the NER pathway. XPB is shown to be a very limited helicase that can act on small DNA bubbles and open a defined region of the DNA duplex. The specialized functions of the accessory domains of XPB are now more clearly delineated. This is also the first direct demonstration of a repair function for archaeal XPB and suggests strongly that the role of XPB in transcription occurred later in evolution than that in repair. [ABSTRACT FROM AUTHOR]

Transcription initiation factor B (TFB) is conserved in eukaryotes and archaea and has an essential role in the recruitment of RNA polymerase to the promoter and the initiation of transcription. The genome of Sulfolobus solfataricus and related crenarchaea contain three paralogues of the tfb gene. Two of them ( tfb1 and tfb2) encode full-length TFB proteins. The third ( tfb3) is significantly shorter than the other two, possessing an N-terminal Zn ribbon domain but lacking the B-finger and DNA binding domains. In S. solfataricus and Sulfolobus acidocaldarius, tfb3 is one of the most highly upregulated transcripts following exposure to UV irradiation. We demonstrate that S. solfataricus TFB3 binds to the RpoK subunit of RNA polymerase, an interaction dependent on the Zn ribbon motif of TFB3. TFB3 can also interact with the ternary complex of TBP and TFB1 bound to a DNA promoter. TFB3 stimulates transcription in vitro from several promoters in the presence of TFB1 and TBP. These observations are consistent with a model whereby TFB3 activates general transcription in trans, via an interaction with RNA polymerase in the pre-initiation complex. This could provide a mechanism for the modulation of transcription initiation in response to environmental stresses, such as DNA damage. [ABSTRACT FROM AUTHOR]

The genomic DNA of all organisms across the three kingdoms of life needs to be compacted and functionally organized. Key players in these processes are DNA supercoiling, macromolecular crowding and architectural proteins that shape DNA by binding to it. The architectural proteins in bacteria, archaea and eukaryotes generally do not exhibit sequence or structural conservation especially across kingdoms. Instead, we propose that they are functionally conserved. Most of these proteins can be classified according to their architectural mode of action: bending, wrapping or bridging DNA. In order for DNA transactions to occur within a compact chromatin context, genome organization cannot be static. Indeed chromosomes are subject to a whole range of remodeling mechanisms. In this review, we discuss the role of (i) DNA supercoiling, (ii) macromolecular crowding and (iii) architectural proteins in genome organization, as well as (iv) mechanisms used to remodel chromosome structure and to modulate genomic activity. We conclude that the underlying mechanisms that shape and remodel genomes are remarkably similar among bacteria, archaea and eukaryotes. [ABSTRACT FROM AUTHOR]

Since the first archaeal genome was sequenced, much attention has been focused on the study of these unique microorganisms. We have learnt that although archaeal DNA metabolic processes (replication, recombination and repair) are more similar to the metabolic processes of Eukarya than those of Bacteria, Archaea are not simply ‘mini Eukarya’. They are, in fact, a mosaic of the eukaryal and bacterial systems that also possess archaeal-specific features. Recent biochemical and structural studies of the proteins that participate in archaeal DNA replication and repair have increased our understanding of these processes. [Copyright &y& Elsevier]

Archaeal DNA repair pathways are not well defined; in particular, there are no convincing candidate proteins for detection of DNA mismatches or the bulky lesions removed by excision repair pathways. Single-stranded DNA-binding proteins (SSBs) play a central role in DNA replication, recombination and repair. The crenarchaeal SSB is a monomer with a single oligonucleotide-binding fold for single-stranded DNA binding coupled to a flexible C-terminal tail reminiscent of bacterial SSB that mediates interactions with other proteins. We demonstrate that Sulfolobus solfataricus SSB can melt DNA containing a mismatch or DNA lesion specifically in vitro. We suggest that a potential role for SSB in archaea is the detection of DNA damage due to local destabilisation of the DNA double helix, followed by recruitment of specific repair proteins. Proteins interacting specifically with a single-stranded DNA:SSB complex include several known or putative DNA repair proteins and DNA helicases. [Copyright &y& Elsevier]

Holliday junction-resolving enzymes are ubiquitous, structure-specific endonucleases that resolve four-way DNA junctions by the introduction of paired nicks in opposing strands, and are required for homologous recombination, double-strand break repair, recombination-dependent restart of stalled or collapsed DNA replication forks, and phage DNA processing. Here, we present the first steady-state kinetic characterisation of a junction-resolving enzyme; the Hje endonuclease from Sulfolobus solfataricus. We demonstrate that substrate turnover by Hje is sequence-independent and limited largely by the rate of cleavage of the phosphodiester bonds of the bound Holliday junction substrate, rather than substrate association or product dissociation. Reaction rates under multiple turnover conditions compare favourably with type II restriction enzymes. These properties, coupled with a high level of specificity for four-way junctions over all other DNA substrates, make Hje a suitable enzyme for applications requiring the detection and cleavage of Holliday junctions in vitro. [Copyright &y& Elsevier]

Structure-specific nucleases of the XPF/Mus81 family function in several DNA recombination and repair pathways in eukaryotes, cleaving a variety of flap and branched DNA substrates. Mus81 and XPF are clearly related evolutionarily but differ markedly in their substrate specificity and protein partners. We demonstrate that the XPF endonuclease from Sulfolobus solfataricus, which is dependent on the sliding clamp proliferating cell nuclear antigen for activity, represents an ancestral form of the XPF/Mus81 family, with key properties in common with both enzymes. The archaeal XPF has a domain organization and sequence preference very similar to eukaryal XPF-ERCC1. However, the archaeal enzyme has a pronounced preference for Mus81-type substrates such as D loops, nicked four-way junctions, and 3' flaps. These all have in common a 5'-DNA end next to the cleavage site. The availability of the sliding clamp proliferating cell nuclear antigen may dictate the activity of Sulfolobus XPF in vivo. [ABSTRACT FROM AUTHOR]

Recently, several advances have been made in the understanding of the form and function of archaeal chromatin. Remarkable parallels can be drawn between the structure and modification of chromatin components in the archaeal and the eukaryotic domains of life. Indeed, it now appears that key components of the hugely complex eukaryotic chromatin regulatory machinery were established before the divergence of the archaeal and eukaryotic lineages. [Copyright &y& Elsevier]

The structure of Escherichia coli cofactor-dependent phosphoglycerate mutase (dPGM), complexed with the potent inhibitor vanadate, has been determined to a resolution of 1.30 A˚ (R-factor 0.159; R-free 0.213). The inhibitor is present in the active site, principally as divanadate, but with evidence of additional vanadate moieties at either end, and representing a different binding mode to that observed in the structural homologue prostatic acid phosphatase. The analysis reveals the enzyme-ligand interactions involved in inhibition of the mutase activity by vanadate and identifies a water molecule, observed in the native E. coli dPGM structure which, once activated by vanadate, may dephosphorylate the active protein. Rather than reflecting the active conformation previously observed for E. coli dPGM, the inhibited protein's conformation resembles that of the inactive dephosphorylated Saccharomyces cerevisiae dPGM. The provision of a high-resolution structure of both active and inactive forms of dPGM from a single organism, in conjunction with computational modelling of substrate molecules in the active site provides insight into the binding of substrates and the specific interactions necessary for three different activities, mutase, synthase and phosphatase, within a single active site. The sequence similarity of E. coli and human dPGMs allows us to correlate stucture with clinical pathology. [Copyright &y& Elsevier]

Junction-resolving enzymes are ubiquitous nucleases that are important for DNA repair and recombination and act on DNA molecules containing branch points, especially four-way junctions. They show a pronounced selectivity for the structure of the DNA substrate but, despite its importance, the structural selectivity is not well understood. This poses an intriguing challenge in molecular recognition on a relatively large scale. [ABSTRACT FROM AUTHOR]

A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised: both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 × 104) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base. [ABSTRACT FROM AUTHOR]

In this issue, show that the CRISPR endonuclease Cas6 wraps an unstructured CRISPR RNA repeat around its surface to achieve recognition and cleavage, suggesting that palindromic RNA is not a feature of many CRISPR subtypes. [ABSTRACT FROM AUTHOR]

Discusses the resolution of the paradox that homologous recombination is a fundamental process present in virtually all life forms and that strand-exchange proteins RecA/Rad51 are universal. Definition of holliday junction-resolving enzymes; Similarity of the pox virus enzymes to the fungal than to the eubacterial enzymes.

The majority of archaeal viral genes are of unknown function hindering our understanding of the virus life cycle and viral interactions with their host. Here, we first describe functional characterization of ORF131b ( gp17 ) and ORF436 ( gp18 ) of Sulfolobus islandicus rod-shaped virus 2 (SIRV2), both encoding proteins of unknown function and forming an operon with ORF207 ( gp19 ). SIRV2 gp17 was found to be a single-stranded DNA (ssDNA) binding protein different in structure from all previously characterized ssDNA binding proteins. Mutagenesis of a few conserved basic residues suggested a U-shaped binding path for ssDNA. The recombinant gp18 showed an ssDNA annealing activity often associated with helicases and recombinases. To gain insight into the biological role of the entire operon, we characterized SIRV2 gp19 and showed it to possess a 5′ → 3′ ssDNA exonuclease activity, in addition to the previously demonstrated ssDNA endonuclease activity. Further, in vitro pull-down assay demonstrated interactions between gp17 and gp18 and between gp18 and gp19 with the former being mediated by the intrinsically disordered C-terminus of gp17. The strand-displacement replication mode proposed previously for rudiviruses and the close interaction among the ssDNA binding, annealing and nuclease proteins strongly point to a role of the gene operon in genome maturation and/or DNA recombination that may function in viral DNA replication/repair. [ABSTRACT FROM AUTHOR]

Viruses infecting hyperthermophilic archaea typically do not encode DNA polymerases, raising questions regarding their genome replication. Here, using a yeast two-hybrid approach, we have assessed interactions between proteins of Sulfolobus islandicus rod-shaped virus 2 (SIRV2) and the host-encoded proliferating cell nuclear antigen (PCNA), a key DNA replication protein in archaea. Five SIRV2 proteins were found to interact with PCNA, providing insights into the recruitment of host replisome for viral DNA replication. [ABSTRACT FROM AUTHOR]

CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as 'interference'. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system. [ABSTRACT FROM AUTHOR]

The Cas4 protein is one of the core CRISPR-associated (Cas) proteins implicated in the prokaryotic CRISPR system for antiviral defence. Cas4 is thought to play a role in the capture of new viral DNA sequences for incorporation into the host genome. No biochemical activity has been reported for Cas4, but it is predicted to include a RecB nuclease domain. We show here that Cas4 family proteins from the archaeon Sulfolobus solfataricus utilise four conserved cysteine residues to bind an iron-sulfur cluster in an arrangement reminiscent of the AddB nuclease of Bacillus subtilis. The Cas4 family protein Sso0001 is a 5' to 3' single stranded DNA exonuclease in vitro that is stalled by extrahelical DNA adducts. A role for Cas4 in DNA duplex strand resectioning to generate recombinogenic 3' single stranded DNA overhangs is proposed. Comparison of the AddB structure with that of a related bacterial nuclease from Eubacterium rectales reveals that the iron-sulfur cluster can be replaced by a zinc ion without disrupting the protein structure, with implications for the evolution of iron-sulfur binding proteins. [ABSTRACT FROM AUTHOR]

Background: The ubiquitous Rad50 and Mre11 proteins play a key role in many processes involved in the maintenance of genome integrity in Bacteria and Eucarya, but their function in the Archaea is presently unknown. We showed previously that in most hyperthermophilic archaea, rad50-mre11 genes are linked to nurA encoding both a single-strand endonuclease and a 5' to 3' exonuclease, and herA, encoding a bipolar DNA helicase which suggests the involvement of the four proteins in common molecular pathway(s). Since genetic tools for hyperthermophilic archaea are just emerging, we utilized immuno-detection approaches to get the first in vivo data on the role(s) of these proteins in the hyperthermophilic crenarchaeon Sulfolobus acidocaldarius. Results: We first showed that S. acidocaldarius can repair DNA damage induced by high doses of gamma rays, and we performed a time course analysis of the total levels and sub-cellular partitioning of Rad50, Mre11, HerA and NurA along with the RadA recombinase in both control and irradiated cells. We found that during the exponential phase, all proteins are synthesized and display constant levels, but that all of them exhibit a different sub-cellular partitioning. Following gamma irradiation, both Mre11 and RadA are immediately recruited to DNA and remain DNAbound in the course of DNA repair. Furthermore, we show by immuno-precipitation assays that Rad50, Mre11 and the HerA helicase interact altogether. Conclusion: Our analyses strongly support that in Sulfolobus acidocaldarius, the Mre11 protein and the RadA recombinase might play an active role in the repair of DNA damage introduced by gamma rays and/or may act as DNA damage sensors. Moreover, our results demonstrate the functional interaction between Mre11, Rad50 and the HerA helicase and suggest that each protein play different roles when acting on its own or in association with its partners. This report provides the first in vivo evidence supporting the implication of the Mre11 protein in DNA repair processes in the Archaea and showing its interaction with both Rad50 and the HerA bipolar helicase. Further studies on the functional interactions between these proteins, the NurA nuclease and the RadA recombinase, will allow us to define their roles and mechanism of action. [ABSTRACT FROM AUTHOR]

Abstract: The resolving enzyme Hjc, which cleaves Holliday junctions with a high degree of structural specificity, is conserved in all archaea. Like RuvC in Escherichia coli, Hjc functions in the related processes of homologous recombination and double-strand break repair. In bacteria, the RuvAB complex binds Holliday junctions and catalyses ATP-dependent branch migration, but the equivalent proteins in archaea and eukarya are unknown. Here, we demonstrate that Hjc from Sulfolobus solfataricus forms a physical interaction with the sliding clamp PCNA via a C-terminal PCNA-interacting peptide (PIP) motif in Hjc. PCNA stimulates the Holliday junction cleavage activity of Hjc in vitro, and deletion of the PIP motif abrogates this effect. This is the first report of a functional interaction between a sliding clamp and a junction-resolving enzyme, and raises the possibility that PCNA could recruit a variety of different proteins to act on Holliday junctions in vivo. [Copyright &y& Elsevier]

Examines the recruitment of intron-encoded and co-opted proteins in splicing of the b13 group I intron RNA. Binding of protein to b13 RNA; Translation of the functional b13 maturase domain; Description of b13 ribonucleoprotein.

Provides information on an experiment investigating the dissection of the sequence specificity of the Holliday junction endonuclease CCE1. Methodology used to conduct the experiment; Systematic investigation of sequence specificity of CCE1; Information on the effect of junction sequence on CCE1 binding affinity; Findings of the experiment.

Transcription factor IIH (TFIIH) plays essential roles in both the initiation of RNA Polymerase II-mediated transcription and the Nucleotide Excision Repair (NER) pathway in eukaryotes. In NER, the 7-subunit TFIIH Core sub-complex is responsible for the opening and extension of the DNA bubble created at the lesion site, utilizing the molecular motors XPB and XPD. Mutations in Core subunits are associated with a series of severe autosomal recessive disorders characterised by symptoms such as mild-to-extreme photosensitivity, premature ageing, physical and neurological anomalies, and in some cases an increased susceptibility to cancer. Although TFIIH Core has been successfully obtained in the past, the process has always remained challenging and laborious, involving many steps that severely hindered the amount of pure, active complex obtained. This has limited biochemical and functional studies of the NER process. Here we describe improved and simplified processes for the cloning, expression and purification of the 7-subunit TFIIH Core sub-complex. The combined use of auto-cleavable 2A-like sequences derived from the Foot-and-Mouth Disease Virus (FMDV) and the MultiBac™ cloning system, a powerful baculoviral expression vector specifically conceived for the obtaining of multi-subunit eukaryotic complexes, allowed us to obtain a single, 7-gene plasmid in a short time using regular restriction cloning strategies. Additionally, expression of the construct in High Five™ insect cells paired with a simple 5-step purification protocol allowed the extraction of a pure, active TFIIH Core sub-complex in milligram quantities. • Auto-cleavable 2A-like sequences effectively simplify the cloning of multi-subunit complexes. • A versatile 6xHis-V5 epitope-TEV cleavable tag facilitates streamlined purification. • 7-subunit, ~350 kDa sub-complex cloned using the MutiBac™ system. • Pure sub-complex obtained in mg quantities using High Five™ insect cells as an expression system. [ABSTRACT FROM AUTHOR]

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix. [ABSTRACT FROM AUTHOR]

Cyclic oligoadenylate (cOA) secondary messengers are generated by type III CRISPR systems in response to viral infection. cOA allosterically activates the CRISPR ancillary ribonucleases Csx1/Csm6, which degrade RNA non-specifically using a HEPN (Higher Eukaryotes and Prokaryotes, Nucleotide binding) active site. This provides effective immunity but can also lead to growth arrest in infected cells, necessitating a means to deactivate the ribonuclease once viral infection has been cleared. In the crenarchaea, dedicated ring nucleases degrade cA 4 (cOA consisting of 4 AMP units), but the equivalent enzyme has not been identified in bacteria. We demonstrate that, in Thermus thermophilus HB8 , the uncharacterized protein TTHB144 is a cA 4 -activated HEPN ribonuclease that also degrades its activator. TTHB144 binds and degrades cA 4 at an N-terminal CARF (CRISPR-associated Rossman fold) domain. The two activities can be separated by site-directed mutagenesis. TTHB144 is thus the first example of a self-limiting CRISPR ribonuclease. Unlabelled Image • TTHB144 is a cyclic tetra-adenylate activated ribonuclease. • TTHB144 degrades cyclic tetra-adenylate at its CARF domain. • Self-limiting enzymes like TTHB144 may regulate anti-viral signaling in bacteria. [ABSTRACT FROM AUTHOR]

Whereas the infection cycles of many bacterial and eukaryotic viruses have been characterized in detail, those of archaeal viruses remain largely unexplored. Recently, studies on a few model archaeal viruses such as SIRV2 (Sulfolobus islandicus rod-shaped virus) have revealed an unusual lysis mechanism that involves the formation of pyramidal egress structures on the host cell surface. To expand understanding of the infection cycle of SIRV2, we aimed to functionally characterize gp1, which is a SIRV2 gene with unknown function. The SIRV2_Gp1 protein is highly expressed during early stages of infection and it is the only protein that is encoded twice on the viral genome. It harbours a helix-turn-helix motif and was therefore hypothesized to bind DNA. The DNA-binding behavior of SIRV2_Gp1 was characterized with electrophoretic mobility shift assays and atomic force microscopy. We provide evidence that the protein interacts with DNA and that it forms large aggregates, thereby causing extreme condensation of the DNA. Furthermore, the N-terminal domain of the protein mediates toxicity to the viral host Sulfolobus. Our findings may lead to biotechnological applications, such as the development of a toxic peptide for the containment of pathogenic bacteria, and add to our understanding of the Rudiviral infection cycle. [ABSTRACT FROM AUTHOR]

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event. [ABSTRACT FROM AUTHOR]

The evolution of CRISPR-cas loci, which encode adaptive immune systems in archaea and bacteria, involves rapid changes, in particular numerous rearrangements of the locus architecture and horizontal transfer of complete loci or individual modules. These dynamics complicate straightforward phylogenetic classification, but here we present an approach combining the analysis of signature protein families and features of the architecture of cas loci that unambiguously partitions most CRISPR-cas loci into distinct classes, types and subtypes. The new classification retains the overall structure of the previous version but is expanded to now encompass two classes, five types and 16 subtypes. The relative stability of the classification suggests that the most prevalent variants of CRISPR-Cas systems are already known. However, the existence of rare, currently unclassifiable variants implies that additional types and subtypes remain to be characterized. [ABSTRACT FROM AUTHOR]

The article discusses a study which shows the sequence specific activity of Cas1 proteins from Escherichia coli and Sulfolobus solfataricus and its role as a determinant of the adaptation process in the adaptive prokaryotic immune system CRISPR-Cas.

CRISPR-Cas is an RNA-mediated adaptive immune system that defends bacteria and archaea against mobile genetic elements. Short mature CRISPR RNAs (crRNAs) are key elements in the interference step of the immune pathway. A CRISPR array composed of a series of repeats interspaced by spacer sequences acquired from invading mobile genomes is transcribed as a precursor crRNA (pre-crRNA) molecule. This pre-crRNA undergoes one or two maturation steps to generate the mature crRNAs that guide CRISPR-associated (Cas) protein(s) to cognate invading genomes for their destruction. Different types of CRISPR-Cas systems have evolved distinct crRNA biogenesis pathways that implicate highly sophisticated processing mechanisms. In Types I and III CRISPR-Cas systems, a specific endoribonuclease of the Cas6 family, either standalone or in a complex with other Cas proteins, cleaves the pre-crRNA within the repeat regions. In Type II systems, the trans-acting small RNA (tracrRNA) base pairs with each repeat of the pre-crRNA to form a dual-RNA that is cleaved by the housekeeping RNase III in the presence of the protein Cas9. In this review, we present a detailed comparative analysis of pre-crRNA recognition and cleavage mechanisms involved in the biogenesis of guide crRNAs in the three CRISPR-Cas types. [ABSTRACT FROM AUTHOR]

Canonical single-stranded DNA-binding proteins (SSBs) from the oligosaccharide/oligonucleotide-binding (OB) domain family are present in all known organisms and are critical for DNA replication, recombination and repair. The SSB from the hyperthermophilic crenarchaeote Sulfolobus solfataricus (SsoSSB) has a 'simple' domain organization consisting of a single DNA-binding OB fold coupled to a flexible C-terminal tail, in contrast with other SSBs in this family that incorporate up to four OB domains. Despite the large differences in the domain organization within the SSB family, the structure of the OB domain is remarkably similar all cellular life forms. However, there are significant differences in the molecular mechanism of ssDNA binding. We have determined the structure of the SsoSSB OB domain bound to ssDNA by NMR spectroscopy. We reveal that ssDNA recognition is modulated by base-stacking of three key aromatic residues, in contrast with the OB domains of human RPA and the recently discovered human homologue of SsoSSB, hSSB1. We also demonstrate that SsoSSB binds ssDNA with a footprint of five bases and with a defined binding polarity. These data elucidate the structural basis of DNA binding and shed light on the molecular mechanism by which these 'simple' SSBs interact with ssDNA. [ABSTRACT FROM AUTHOR]

The protein Hjc from the thermophilic archaeon Sulfolobus solfataricus (Ss) presented many challenges to both structure solution and formation of stable complexes with its substrate, the DNA four-way or Holliday junction. As the challenges were caused by an uncharacteristically high propensity for rapid and promiscuous crystallisation, we investigated the molecular cause of this behaviour, corrected it by mutagenesis, and solved the X-ray crystal structures of the two mutants. An active site mutant SsHjcA32A crystallised in space group I23 (a 144.2 Å; 68 % solvent), and a deletion of a key crystal contact site, SsHjcδ62-63 crystallised in space group P21 (a 64.60, b 61.83, c 55.25 Å; β = 95.74°; 28 % solvent). Characterisation and comparative analysis of the structures are presented along with discussion of the pitfalls of the use of protein engineering to alter crystallisability while maintaining biological function. [ABSTRACT FROM AUTHOR]

Summary: The Clustered Regularly Interspaced Palindromic Repeats (CRISPR) system is an adaptive immune system in prokaryotes. Interference complexes encoded by CRISPR-associated (cas) genes utilize small RNAs for homology-directed detection and subsequent degradation of invading genetic elements, and they have been classified into three main types (I–III). Type III complexes share the Cas10 subunit but are subclassifed as type IIIA (CSM) and type IIIB (CMR), depending on their specificity for DNA or RNA targets, respectively. The role of CSM in limiting the spread of conjugative plasmids in Staphylococcus epidermidis was first described in 2008. Here, we report a detailed investigation of the composition and structure of the CSM complex from the archaeon Sulfolobus solfataricus, using a combination of electron microscopy, mass spectrometry, and deep sequencing. This reveals a three-dimensional model for the CSM complex that includes a helical component strikingly reminiscent of the backbone structure of the type I (Cascade) family. [Copyright &y& Elsevier]

The competition between viruses and hosts is played out in all branches of life. Many prokaryotes have an adaptive immune system termed 'CRISPR' (clustered regularly interspaced short palindromic repeats) which is based on the capture of short pieces of viral DNA. The captured DNA is integrated into the genomic DNA of the organism flanked by direct repeats, transcribed and processed to generate crRNA (CRISPR RNA) that is loaded into a variety of effector complexes. These complexes carry out sequence-specific detection and destruction of invading mobile genetic elements. In the present paper, we report the structure and activity of a Cas6 (CRISPR-associated 6) enzyme (Sso1437) from Sulfolobus solfataricus responsible for the generation of unitlength crRNA species. The crystal structure reveals an unusual dimeric organization that is important for the enzyme's activity. In addition, the active site lacks the canonical catalytic histidine residue that has been viewed as an essential feature of the Cas6 family. Although several residues contribute towards catalysis, none is absolutely essential. Coupled with the very low catalytic rate constants of the Cas6 family and the plasticity of the active site, this suggests that the crRNA recognition and chaperone-like activities of the Cas6 family should be considered as equal to or even more important than their role as traditional enzymes. [ABSTRACT FROM AUTHOR]

DinG (damage inducible gene G) is a bacterial superfamily 2 helicase with 5′→3′ polarity. DinG is related to the XPD (xeroderma pigmentosum complementation group D) helicase family, and they have in common an FeS (iron–sulfur)-binding domain that is essential for the helicase activity. In the bacilli and clostridia, the DinG helicase has become fused with an N-terminal domain that is predicted to be an exonuclease. In the present paper we show that the DinG protein from Staphylococcus aureus lacks an FeS domain and is not a DNA helicase, although it retains DNA-dependent ATP hydrolysis activity. Instead, the enzyme is an active 3′→5′ exonuclease acting on single-stranded DNA and RNA substrates. The nuclease activity can be modulated by mutation of the ATP-binding cleft of the helicase domain, and is inhibited by ATP or ADP, suggesting a modified role for the inactive helicase domain in the control of the nuclease activity. By degrading rather than displacing RNA or DNA strands, the S. aureus DinG nuclease may accomplish the same function as the canonical DinG helicase. [ABSTRACT FROM AUTHOR]

Summary: The prokaryotic clusters of regularly interspaced palindromic repeats (CRISPR) system utilizes genomically encoded CRISPR RNA (crRNA), derived from invading viruses and incorporated into ribonucleoprotein complexes with CRISPR-associated (CAS) proteins, to target and degrade viral DNA or RNA on subsequent infection. RNA is targeted by the CMR complex. In Sulfolobus solfataricus, this complex is composed of seven CAS protein subunits (Cmr1-7) and carries a diverse “payload” of targeting crRNA. The crystal structure of Cmr7 and low-resolution structure of the complex are presented. S. solfataricus CMR cleaves RNA targets in an endonucleolytic reaction at UA dinucleotides. This activity is dependent on the 8 nt repeat-derived 5′ sequence in the crRNA, but not on the presence of a protospacer-associated motif (PAM) in the target. Both target and guide RNAs can be cleaved, although a single molecule of guide RNA can support the degradation of multiple targets. [ABSTRACT FROM AUTHOR]

In response to viral infection, many prokaryotes incorporate fragments of virus-derived DNA into loci called clustered regularly interspaced short palindromic repeats (CRISPRs). The loci are then transcribed, and the processed CRISPR transcripts are used to target invading viral DNA and RNA. The Escherichia coli "CRISPR-associated complex for antiviral defense" (CASCADE) is central in targeting invading DNA. Here we report the structural and functional characterization of an archaeal CASCADE (aCASCADE) from Sulfolobus solfataricus. Tagged Csa2 (Cas7) expressed in S. solfataricus co-purifies with Cas5a-, Cas6-, Csa5-, and Cas6-processed CRISPR-RNA (crRNA). Csa2, the dominant protein in aCASCADE, forms a stable complex with Cas5a. Transmission electron microscopy reveals a helical complex of variable length, perhaps due to substoichiometric amounts of other CASCADE components. A recombinant Csa2-Cas5a complex is sufficient to bind crRNA and complementary ssDNA. The structure of Csa2 reveals a crescent-shaped structure unexpectedly composed of a modified RNA-recognition motif and two additional domains present as insertions in the RNA-recognition motif. Conserved residues indicate potential crRNA- and target DNA-binding sites, and the H160A variant shows significantly reduced affinity for crRNA. We propose a general subunit architecture for CASCADE in other bacteria and Archaea. [ABSTRACT FROM AUTHOR]

Abstract: DNA recombinases (RecA in bacteria, Rad51 in eukarya and RadA in archaea) catalyse strand exchange between homologous DNA molecules, the central reaction of homologous recombination, and are among the most conserved DNA repair proteins known. RecA is the sole protein responsible for this reaction in bacteria, whereas there are several Rad51 paralogs that cooperate to catalyse strand exchange in eukaryotes. All archaea have at least one (and as many as four) RadA paralog, but their function remains unclear. Herein, we show that the three RadA paralogs encoded by the Sulfolobus solfataricus genome are expressed under normal growth conditions and are not UV inducible. We demonstrate that one of these proteins, Sso2452, which is representative of the large archaeal RadC subfamily of archaeal RadA paralogs, functions as an ATPase that binds tightly to single-stranded DNA. However, Sso2452 is not an active recombinase in vitro and inhibits D-loop formation by RadA. We present the high-resolution crystal structure of Sso2452, which reveals key structural differences from the canonical RecA family recombinases that may explain its functional properties. The possible roles of the archaeal RadA paralogs in vivo are discussed. [Copyright &y& Elsevier]

Bacterial pathogens need to scavenge iron from their host for growth and proliferation during infection. They have evolved several strategies to do this, one being the biosynthesis and excretion of small, high-affinity iron chelators known as siderophores. The biosynthesis of siderophores is an important area of study, not only for potential therapeutic intervention but also to illuminate new enzyme chemistries. Two general pathways for siderophore biosynthesis exist: the well-characterized nonribosomal peptide synthetase (NRPS)-dependent pathway and the NRPS-independent siderophore (NIS) pathway, which relies on a different family of sparsely investigated synthetases. Here we report structural and biochemical studies of AcsD from Pectobacterium (formerly Erwinia) chrysanthemi, an NIS synthetase involved in achromobactin biosynthesis. The structures of ATP and citrate complexes provide a mechanistic rationale for stereospecific formation of an enzyme-bound (3R)-citryladenylate, which reacts with L-serine to form a likely achromobactin precursor. AcsD is a unique acyladenylate-forming enzyme with a new fold and chemical catalysis strategy. [ABSTRACT FROM AUTHOR]

AcsD, a type A siderophore synthetase with a molecular weight of 71 140 Da from Pectobacterium chrysanthemi, has been expressed, purified and crystallized at 293 K. The protein crystallized in the primitive orthorhombic space group P212121, with unit-cell parameters a = 80.3, b = 95.7, c = 161.1 Å, α = β = γ = 90°. Systematic absences were consistent with space group P212121. A complete data set has been collected to 2.25 Å resolution on BM14 at the ESRF. Consideration of the likely solvent content suggested that the asymmetric unit contained two molecules. Gel-filtration experiments indicated that the protein was a dimer, although self-rotation analyses did not detect a convincing twofold symmetry axis in the asymmetric unit. The protein has no convincing sequence match to any known structure and thus solution is likely to require experimental phasing. [ABSTRACT FROM AUTHOR]

Summary: The XPD helicase (Rad3 in Saccharomyces cerevisiae) is a component of transcription factor IIH (TFIIH), which functions in transcription initiation and Nucleotide Excision Repair in eukaryotes, catalyzing DNA duplex opening localized to the transcription start site or site of DNA damage, respectively. XPD has a 5′ to 3′ polarity and the helicase activity is dependent on an iron-sulfur cluster binding domain, a feature that is conserved in related helicases such as FancJ. The xpd gene is the target of mutation in patients with xeroderma pigmentosum, trichothiodystrophy, and Cockayne''s syndrome, characterized by a wide spectrum of symptoms ranging from cancer susceptibility to neurological and developmental defects. The 2.25 Å crystal structure of XPD from the crenarchaeon Sulfolobus tokodaii, presented here together with detailed biochemical analyses, allows a molecular understanding of the structural basis for helicase activity and explains the phenotypes of xpd mutations in humans. [Copyright &y& Elsevier]

Single-strand DNA (ssDNA)-binding proteins (SSBs) are ubiquitous and essential for a wide variety of DNA metabolic processes, including DNA replication, recombination, DNA damage detection and repair. SSBs have multiple roles in binding and sequestering ssDNA, detecting DNA damage, stimulating nucleases, helicases and strand-exchange proteins, activating transcription and mediating protein–protein interactions. In eukaryotes, the major SSB, replication protein A (RPA), is a heterotrimer. Here we describe a second human SSB (hSSB1), with a domain organization closer to the archaeal SSB than to RPA. Ataxia telangiectasia mutated (ATM) kinase phosphorylates hSSB1 in response to DNA double-strand breaks (DSBs). This phosphorylation event is required for DNA damage-induced stabilization of hSSB1. Upon induction of DNA damage, hSSB1 accumulates in the nucleus and forms distinct foci independent of cell-cycle phase. These foci co-localize with other known repair proteins. In contrast to RPA, hSSB1 does not localize to replication foci in S-phase cells and hSSB1 deficiency does not influence S-phase progression. Depletion of hSSB1 abrogates the cellular response to DSBs, including activation of ATM and phosphorylation of ATM targets after ionizing radiation. Cells deficient in hSSB1 exhibit increased radiosensitivity, defective checkpoint activation and enhanced genomic instability coupled with a diminished capacity for DNA repair. These findings establish that hSSB1 influences diverse endpoints in the cellular DNA damage response. [ABSTRACT FROM AUTHOR]

Hel308 is a superfamily 2 helicase conserved in eukaryotes and archaea. It is thought to function in the early stages of recombination following replication fork arrest and has a specificity for removal of the lagging strand in model replication forks. A homologous helicase constitutes the N-terminal domain of human DNA polymerase Q. The Drosophila homologue mus301 is implicated in double strand break repair and meiotic recombination. We have solved the high resolution crystal structure of Hel308 from the crenarchaeon Sulfolobus solfataricus, revealing a five-domain structure with a central pore lined with essential DNA binding residues. The fifth domain is shown to act as an autoinhibitory domain or molecular brake, clamping the single-stranded DNA extruded through the central pore of the helicase structure to limit the helicase activity of the enzyme. This provides an elegant mechanism to tune the processivity of the enzyme to its functional role. Hel308 can displace streptavidin from a biotinylated DNA molecule, and this activity is only partially inhibited when the DNA is pre-bound with abundant DNA-binding proteins RPA or Alba1, whereas pre-binding with the recombinase RadA has no effect on activity. These data suggest that one function of the enzyme may be in the removal of bound proteins at stalled replication forks and recombination intermediates. [ABSTRACT FROM AUTHOR]

Abstract: XPB is a superfamily 2 helicase with a 3′–5′ polarity. In eukaryotes, XPB is an integral subunit of the transcription factor TFIIH, which plays a dual role in DNA opening at RNA polymerase II promoters and in establishing the repair bubble around a DNA lesion in nucleotide excision repair. Eukaryotic XPB has only very limited helicase activity in vitro and may function as a DNA-dependent molecular switch to catalyse local distortion of DNA in transcription and repair. Most archaea have one or two homologues of the XPB protein with a presumed role in DNA repair, but only one other subunit of the TFIIH complex, the 5′–3′ helicase XPD, has been identified in archaea. Here we report the biochemical characterisation of the two homologous XPB proteins from the crenarchaeon Sulfolobus solfataricus. Although both proteins are single-stranded-DNA-stimulated ATPases, neither displays any helicase activity in vitro, consistent with recent studies of eukaryotic XPB. In almost all archaeal genomes, the xpb gene lies adjacent to a conserved partner gene, and we demonstrate that these two gene products form a physical interaction in vitro. We propose the name Bax1 (Binds archaeal XPB) for this protein, which has a predicted endonuclease domain. XPB and Bax1 may collaborate in processing nucleic acid in an archaeal-specific DNA repair pathway. [Copyright &y& Elsevier]

N-terminal protein acetylation is common in eukaryotes and halophilic archaea, but very rare in bacteria. We demonstrate that some of the most abundant proteins present in the crenarchaeote Sulfolobus solfataricus, including subunits of the thermosome, proteosome and ribosome, are acetylated at the N-terminus. Modification was observed at the N-terminal residues serine, alanine, threonine and methionine-glutamate. A conserved archaeal protein, ssArd1, was cloned and expressed in Escherichia coli, and shown to acetylate the same N-terminal sequences in vitro. The specific activity of ssArd1 is sensitive to protein structure in addition to sequence context. The crenarchaeota and euryarchaeota apparently differ in respect of the frequency of acetylation of Met–Glu termini, which appears much more common in S. solfataricus. This sequence is acetylated by the related Nat3 acetylase in eukarya. ssArd1 thus has a relaxed sequence specificity compared with the eukaryotic N-acetyl transferases, and may represent an ancestral form of the enzyme. This represents another example where archaeal molecular biology resembles that in eukaryotes rather than bacteria. [ABSTRACT FROM AUTHOR]