Archaeal Viruses Research Articles

The SSV1 viral integrase is not essential

Viral integration is a widely conserved characteristic in viruses in all domains of life; however, its necessity is not well understood in many cases. Integration using tyrosine recombinases is one of the most widespread and best characterized mechanisms of integration. We completely removed the tyrosine recombinase integrase from the hyperthermophilic and acidophilic archaeal virus SSV1 using a novel LIPCR technique and found that the virus still replicated and spread in its host Sulfolobus solfataricus without integration. The mutant virus maintained a persistent infection but the integrase-lacking virus was less competitive than the wild-type virus when co-cultured. Based on these results, we discuss the necessity of integration and the possible advantages of this type of replication strategy.

Viruses of the Archaea: a unifying view

DNA viruses of the Archaea have highly diverse and often exceptionally complex morphotypes. Many have been isolated from geothermally heated hot environments, raising intriguing questions about their origins, and contradicting the widespread notion of limited biodiversity in extreme environments. Here, we provide a unifying view on archaeal viruses, and present them as a particular assemblage that is fundamentally different in morphotype and genome from the DNA viruses of the other two domains of life, the Bacteria and Eukarya.

Quantitative dissociation of archaeal virus SH1 reveals distinct capsid proteins and a lipid core

Viruses infecting archaeal cells are less well understood than those infecting eukaryotic and bacterial cells. Here we study the distribution of the structural proteins between the capsid and the membrane of icosahedral SH1 virus, an archaeal virus infecting extreme halophilic Haloarcula hispanica cells. General features such as morphology, linear dsDNA genome and presence of lipids suggest that it may belong to the recently proposed PRD1-adenovirus lineage of viruses. To investigate this we have initiated structural studies of the virion. Quantitative dissociation of SH1 by 3 M urea or by lowering the salt concentration identified a number of soluble capsid-associated proteins (VP2, VP3, VP4, VP6, VP7 and VP9). These released proteins left behind a particle, or lipid core, containing two major proteins VP10 and VP12 and viral phospholipids. VP1 was released from the lipid core in low ionic strength conditions but not with 3 M urea. Approximately half of the protein VP5 stayed with the lipid core and the other half was released. Analysis of the soluble capsid-associated proteins by their sedimentation and hydrodynamic properties suggests that the most abundant proteins, putative capsomers VP4 and VP7, form an intricate pattern of protein complexes. We also observed large differences in the sizes of the complexes determined by the two different methods suggesting an elongated overall structure for most of the capsid-associated proteins or protein complexes. This work verifies that there is an internal membrane vesicle residing inside the complex icosahedral capsid that is akin to the overall structure of PRD1-like viruses.

A novel archaeal regulatory protein, Sta1, activates transcription from viral promoters

While studying gene expression of the rudivirus SIRV1 in cells of its host, the hyperthermophilic crenarchaeon Sulfolobus, a novel archaeal transcriptional regulator was isolated. The 14 kDa protein, termed Sulfolobus transcription activator 1, Sta1, is encoded on the host chromosome. Its activating effect on transcription initiation from viral promoters was demonstrated in in vitro transcription experiments using a reconstituted host system containing the RNA polymerase, TATA-binding protein (TBP) and transcription factor B (TFB). Most pronounced activation was observed at low concentrations of either of the two transcription factors, TBP or TFB. Sta1 was able to bind viral promoters independently of any component of the host pre-initiation complex. Two binding sites were revealed by footprinting, one located in the core promoter region and the second ∼30 bp upstream of it. Comparative modeling, NMR and circular dichroism of Sta1 indicated that the protein contained a winged helix–turn–helix motif, most probably involved in DNA binding. This strategy of the archaeal virus to co-opt a host cell regulator to promote transcription of its genes resembles eukaryal virus–host relationships.

Characterization of the Archaeal ThermophileSulfolobusTurreted Icosahedral Virus Validates an Evolutionary Link among Double-Stranded DNA Viruses from All Domains of Life

Icosahedral nontailed double-stranded DNA (dsDNA) viruses are present in all three domains of life, leading to speculation about a common viral ancestor that predates the divergence of Eukarya, Bacteria, and Archaea. This suggestion is supported by the shared general architecture of this group of viruses and the common fold of their major capsid protein. However, limited information on the diversity and replication of archaeal viruses, in general, has hampered further analysis. Sulfolobus turreted icosahedral virus (STIV), isolated from a hot spring in Yellowstone National Park, was the first icosahedral virus with an archaeal host to be described. Here we present a detailed characterization of the components forming this unusual virus. Using a proteomics-based approach, we identified nine viral and two host proteins from purified STIV particles. Interestingly, one of the viral proteins originates from a reading frame lacking a consensus start site. The major capsid protein (B345) was found to be glycosylated, implying a strong similarity to proteins from other dsDNA viruses. Sequence analysis and structural predication of virion-associated viral proteins suggest that they may have roles in DNA packaging, penton formation, and protein-protein interaction. The presence of an internal lipid layer containing acidic tetraether lipids has also been confirmed. The previously presented structural models in conjunction with the protein, lipid, and carbohydrate information reported here reveal that STIV is strikingly similar to viruses associated with the Bacteria and Eukarya domains of life, further strengthening the hypothesis for a common ancestor of this group of dsDNA viruses from all domains of life.

Hot crenarchaeal viruses reveal deep evolutionary connections

The discovery of archaeal viruses provides insights into the fundamental biochemistry and evolution of the Archaea. Recent studies have identified a wide diversity of archaeal viruses within the hot springs of Yellowstone National Park and other high-temperature environments worldwide. These viruses are often morphologically unique and code for genes with little similarity to other known genes in the biosphere, a characteristic that has complicated efforts to trace their evolutionary history. Comparative genomics combined with structural analysis indicate that spindle-shaped virus lineages might be unique to the Archaea, whereas other icosahedral viruses might share a common lineage with viruses of Bacteria and Eukarya. These studies provide insights into the evolutionary history of viruses in all three domains of life.

Structural and Genomic Properties of the Hyperthermophilic Archaeal Virus ATV with an Extracellular Stage of the Reproductive Cycle

A novel virus, ATV, of the hyperthermophilic archaeal genus Acidianus has the unique property of undergoing a major morphological development outside of, and independently of, the host cell. Virions are extruded from host cells as lemon-shaped tail-less particles, after which they develop long tails at each pointed end, at temperatures close to that of the natural habitat, 85 °C. The extracellularly developed tails constitute tubes, which terminate in an anchor-like structure that is not observed in the tail-less particles. A thin filament is located within the tube, which exhibits a periodic structure. Tail development produces a one half reduction in the volume of the virion, concurrent with a slight expansion of the virion surface. The circular, double-stranded DNA genome contains 62,730 bp and is exceptional for a crenarchaeal virus in that it carries four putative transposable elements as well as genes, which previously have been associated only with archaeal self-transmissable plasmids. In total, it encodes 72 predicted proteins, including 11 structural proteins with molecular masses in the range of 12 to 90 kDa. Several of the larger proteins are rich in coiled coil and/or low complexity sequence domains, which are unusual for archaea. One protein, in particular P800, resembles an intermediate filament protein in its structural properties. It is modified in the two-tailed, but not in the tail-less, virion particles and it may contribute to viral tail development. Exceptionally for a crenarchaeal virus, infection with ATV results either in viral replication and subsequent cell lysis or in conversion of the infected cell to a lysogen. The lysogenic cycle involves integration of the viral genome into the host chromosome, probably facilitated by the virus-encoded integrase and this process can be interrupted by different stress factors.

His1 and His2 are distantly related, spindle-shaped haloviruses belonging to the novel virus group, Salterprovirus

Spindle-shaped viruses are a dominant morphotype in hypersaline waters but their molecular characteristics and their relationship to other archaeal viruses have not been determined. Here, we describe the isolation, characteristics and genome sequence of His2, a spindle-shaped halovirus, and compare it to the previously reported halovirus His1. Their particle dimensions, host-ranges and buoyant densities were found to be similar but they differed in their stabilities to raised temperature, low salinity and chloroform. The genomes of both viruses were linear dsDNA, of similar size (His1, 14,464 bp; His2, 16,067 bp) and mol% G + C (∼40%), with long, inverted terminal repeat sequences. The genomic termini of both viruses are likely to possess bound proteins. They shared little nucleotide similarity and, except for their putative DNA polymerase ORFs, no significant similarity at the predicted protein level. A few of the 35 predicted ORFs of both viruses showed significant matches to sequences in GenBank, and these were always to proteins of haloarchaea. Their DNA polymerases showed 42% aa identity, and belonged to the type B group of replicases that use protein-priming. Purified His2 particles were composed of four main proteins (62, 36, 28 and 21 kDa) and the gene for the major capsid protein was identified. Hypothetical proteins similar to His2 VP1 are present in four haloarchaeal genomes but are not part of complete prophages. This, and other evidence, suggests a high frequency of recombination between haloviruses and their hosts. His1 and His2 are unlike fuselloviruses and have been placed in a new virus group, Salterprovirus.

Evolutionary genomics of archaeal viruses: Unique viral genomes in the third domain of life

In terms of virion morphology, the known viruses of archaea fall into two distinct classes: viruses of mesophilic and moderately thermophilic Eueryarchaeota closely resemble head-and-tail bacteriophages whereas viruses of hyperthermophilic Crenarchaeota show a variety of unique morphotypes. In accord with this distinction, the sequenced genomes of euryarchaeal viruses encode many proteins homologous to bacteriophage capsid proteins. In contrast, initial analysis of the crenarchaeal viral genomes revealed no relationships with bacteriophages and, generally, very few proteins with detectable homologs. Here we describe a re-analysis of the proteins encoded by archaeal viruses, with an emphasis on comparative genomics of the unique viruses of Crenarchaeota. Detailed examination of conserved domains and motifs uncovered a significant number of previously unnoticed homologous relationships among the proteins of crenarchaeal viruses and between viral proteins and those from cellular life forms and allowed functional predictions for some of these conserved genes. A small pool of genes is shared by overlapping subsets of crenarchaeal viruses, in a general analogy with the metagenome structure of bacteriophages. The proteins encoded by the genes belonging to this pool include predicted transcription regulators, ATPases implicated in viral DNA replication and packaging, enzymes of DNA precursor metabolism, RNA modification enzymes, and glycosylases. In addition, each of the crenarchaeal viruses encodes several proteins with prokaryotic but not viral homologs, some of which, predictably, seem to have been scavenged from the crenarchaeal hosts, but others might have been acquired from bacteria. We conclude that crenarchaeal viruses are, in general, evolutionarily unrelated to other known viruses and, probably, evolved via independent accretion of genes derived from the hosts and, through more complex routes of horizontal gene transfer, from other prokaryotes.

The ancient Virus World and evolution of cells

BackgroundRecent advances in genomics of viruses and cellular life forms have greatly stimulated interest in the origins and evolution of viruses and, for the first time, offer an opportunity for a data-driven exploration of the deepest roots of viruses. Here we briefly review the current views of virus evolution and propose a new, coherent scenario that appears to be best compatible with comparative-genomic data and is naturally linked to models of cellular evolution that, from independent considerations, seem to be the most parsimonious among the existing ones.ResultsSeveral genes coding for key proteins involved in viral replication and morphogenesis as well as the major capsid protein of icosahedral virions are shared by many groups of RNA and DNA viruses but are missing in cellular life forms. On the basis of this key observation and the data on extensive genetic exchange between diverse viruses, we propose the concept of the ancient virus world. The virus world is construed as a distinct contingent of viral genes that continuously retained its identity throughout the entire history of life. Under this concept, the principal lineages of viruses and related selfish agents emerged from the primordial pool of primitive genetic elements, the ancestors of both cellular and viral genes. Thus, notwithstanding the numerous gene exchanges and acquisitions attributed to later stages of evolution, most, if not all, modern viruses and other selfish agents are inferred to descend from elements that belonged to the primordial genetic pool. In this pool, RNA viruses would evolve first, followed by retroid elements, and DNA viruses. The Virus World concept is predicated on a model of early evolution whereby emergence of substantial genetic diversity antedates the advent of full-fledged cells, allowing for extensive gene mixing at this early stage of evolution. We outline a scenario of the origin of the main classes of viruses in conjunction with a specific model of precellular evolution under which the primordial gene pool dwelled in a network of inorganic compartments. Somewhat paradoxically, under this scenario, we surmise that selfish genetic elements ancestral to viruses evolved prior to typical cells, to become intracellular parasites once bacteria and archaea arrived at the scene. Selection against excessively aggressive parasites that would kill off the host ensembles of genetic elements would lead to early evolution of temperate virus-like agents and primitive defense mechanisms, possibly, based on the RNA interference principle. The emergence of the eukaryotic cell is construed as the second melting pot of virus evolution from which the major groups of eukaryotic viruses originated as a result of extensive recombination of genes from various bacteriophages, archaeal viruses, plasmids, and the evolving eukaryotic genomes. Again, this vision is predicated on a specific model of the emergence of eukaryotic cell under which archaeo-bacterial symbiosis was the starting point of eukaryogenesis, a scenario that appears to be best compatible with the data.ConclusionThe existence of several genes that are central to virus replication and structure, are shared by a broad variety of viruses but are missing from cellular genomes (virus hallmark genes) suggests the model of an ancient virus world, a flow of virus-specific genes that went uninterrupted from the precellular stage of life's evolution to this day. This concept is tightly linked to two key conjectures on evolution of cells: existence of a complex, precellular, compartmentalized but extensively mixing and recombining pool of genes, and origin of the eukaryotic cell by archaeo-bacterial fusion. The virus world concept and these models of major transitions in the evolution of cells provide complementary pieces of an emerging coherent picture of life's history.ReviewersW. Ford Doolittle, J. Peter Gogarten, and Arcady Mushegian.

More barrels from the viral tree of life

There is now unmistakable evidence for a revolution in our comprehension of the viral world. Despite their importance for disease and the insights obtained by studying their fascinating intricacy, viral relationships have long been unclear. The article by Khayat et al. (1) in a recent issue of PNAS marked a milestone in progress. Viral origins have been masked by their diversity and complexity, the way they were studied, and their small size. Viruses evolved cunning solutions to the problems of packing information into genomes of limited size and to entering and exploiting their host. Until fairly recently, each family was studied separately by its own group of scientific admirers. Structurally, viruses could only be imaged by low-resolution electron microscopy (EM), with most too large to tackle with crystallography. Advances in imaging techniques, changes in the way that viruses are studied, and increasing structural evidence all are contributing to major change. There is now excellent evidence to support the idea that viruses fall into lineages identifiable by features in common with ancient precursors (2). The best-supported lineage has “double-barrel trimer” coat proteins and icosahedral virions. Its members infect Gram-positive and Gram-negative bacteria, as well as animals, and include very large viruses infecting insects, algae, and amoebae. Structures are at hand for coat proteins of viruses infecting animals, bacteria, and algae, but that for an archaeal virus was lacking. This crucial missing link is found in the thermophilic archaeal Sulfolobus turreted icosahedral virus (STIV) (1). We now have solid structural evidence that at least one lineage originated billions of years ago, before life separated into its three domains: Bacteria, Archaea, and Eukarya. To appreciate this achievement, one must recognize the dramatic progress in describing viral architecture since the first crystal structure of a complete viral particle, or virion, appeared (3). This plant …

Structure of an archaeal virus capsid protein reveals a common ancestry to eukaryotic and bacterial viruses.

Archaea and their viruses are poorly understood when compared with the Eukarya and Bacteria domains of life. We report here the crystal structure of the major capsid protein (MCP) of the Sulfolobus turreted icosahedral virus, an archaeal virus isolated from an acidic hot spring (pH 2-4, 72-92 degrees C) in Yellowstone National Park. The structure is nearly identical to the MCP structures of the eukaryotic Paramecium bursaria Chlorella virus, and the bacteriophage PRD1, and shows a common fold with the mammalian adenovirus. Structural analysis of the capsid architecture, determined by fitting the subunit into the electron cryomicroscopy reconstruction of the virus, identified a number of key interactions that are akin to those observed in adenovirus and PRD1. The similar capsid proteins and capsid architectures strongly suggest that these viral capsids originated and evolved from a common ancestor. Hence, this work provides a previously undescribed example of a viral relationship spanning the three domains of life (Eukarya, Bacteria, and Archaea). The MCP structure also provides insights into the stabilizing forces required for extracellular hyperthermophilic proteins to tolerate high-temperature hot springs.

Constituents of SH1, a Novel Lipid-Containing Virus Infecting the Halophilic Euryarchaeon Haloarcula hispanica

Recent studies have indicated that a number of bacterial and eukaryotic viruses that share a common architectural principle are related, leading to the proposal of an early common ancestor. A prediction of this model would be the discovery of similar viruses that infect archaeal hosts. Our main interest lies in icosahedral double-stranded DNA (dsDNA) viruses with an internal membrane, and we now extend our studies to include viruses infecting archaeal hosts. While the number of sequenced archaeal viruses is increasing, very little sequence similarity has been detected between bacterial and eukaryotic viruses. In this investigation we rigorously show that SH1, an icosahedral dsDNA virus infecting Haloarcula hispanica, possesses lipid structural components that are selectively acquired from the host pool. We also determined the sequence of the 31-kb SH1 genome and positively identified genes for 11 structural proteins, with putative identification of three additional proteins. The SH1 genome is unique and, except for a few open reading frames, shows no detectable similarity to other published sequences, but the overall structure of the SH1 virion and its linear genome with inverted terminal repeats is reminiscent of lipid-containing dsDNA bacteriophages like PRD1.

Purification, crystallization and preliminary X-ray diffraction studies of the archaeal virus resolvase SIRV2

The Holliday junction (or four-way junction) is the universal DNA intermediate whose interaction with resolving proteins is one of the major events in the recombinational process. These proteins, called DNA junction-resolving enzymes or resolvases, bind to the junction and catalyse DNA cleavage, promoting the release of two DNA duplexes. SIRV2 Hjc, a viral resolvase infecting a thermophylic archaeon, has been cloned, expressed and purified. Crystals have been obtained in space group C2, with unit-cell parameters a = 147.8, b = 99.9, c = 87.6, beta = 109.46 degrees, and a full data set has been collected at 3.4 A resolution. The self-rotation function indicates the presence of two dimers in the asymmetric unit and a high solvent content (77%). Molecular-replacement trials using known similar resolvase structures have so far been unsuccessful, indicating possible significant structural rearrangements.

In vitro DNA Packaging of PRD1: A Common Mechanism for Internal-membrane Viruses

PRD1 is the type virus of the Tectiviridae family. Its linear double-stranded DNA genome has covalently attached terminal proteins and is surrounded by a membrane, which is further enclosed within an icosahedral protein capsid. Similar to tailed bacteriophages, PRD1 packages its DNA into a preformed procapsid. The PRD1 putative packaging ATPase P9 is a structural protein located at a unique vertex of the capsid. An in vitro system for packaging DNA into preformed empty procapsids was developed. The system uses cell extracts of overexpressed P9 protein and empty procapsids from a P9-deficient mutant virus infection and PRD1 DNA containing a LacZalpha-insert. The in vitro packaged virions produce distinctly blue plaques when plated on a suitable host. This is the first time that a viral genome is packaged in vitro into a membrane vesicle. Comparison of PRD1 P9 with putative packaging ATPase sequences from bacterial, archaeal and eukaryotic viruses revealed a new packaging ATPase-specific motif. Surprisingly the viruses having this packaging ATPase motif, and thus considered to be related, were the same as those recently grouped together using the coat protein fold and virion architecture. Our finding here strongly supports the idea that all these viruses infecting hosts in all domains of life had a common ancestor.

Mitigation strategies to reduce enteric methane emissions from dairy cows: Update review

Enteric methane (CH4) emission is a major contributor to Canadian greenhouse gas emissions, and also a loss of feed energy during production. The objective of this paper is to provide an update on current management practices and new dietary strategies recently proposed to reduce CH4 emissions from ruminants. Existing mitigation strategies for dairy, e.g., the addition of ionophores, fats, use of high-quality forages, and increased use of grains, have been well researched and applied. These nutritional changes reduce CH4 emissions by manipulating ruminal fermentation, directly inhibiting methanogens and protozoa, or by diverting hydrogen ions away from methanogens. Current literature has identified new CH4 mitigation options. These include the addition of probiotics, acetogens, bacteriocins, archaeal viruses, organic acids, plant extracts (e.g., essential oils) to the diet, as well as immunization, and genetic selection of cows. These new strategies are promising, but more research is needed to validate these approaches and to assess in vivo their effectiveness in reducing CH4 production by dairy cows. It is also important to evaluate CH4 mitigation strategies in terms of the total greenhouse gas budget and to consider the cost associated with the various strategies. More basic understanding of the natural differences in digestion efficiencies among animals as well as a better knowledge of methanogens and their interaction with other organisms in the rumen would enable us to exploit the potential of some of the new CH4 mitigation strategies for dairy cattle production. Key words: Enteric methane, dairy cattle, mitigation

The structure of a thermophilic archaeal virus shows a double-stranded DNA viral capsid type that spans all domains of life.

Of the three domains of life (Eukarya, Bacteria, and Archaea), the least understood is Archaea and its associated viruses. Many Archaea are extremophiles, with species that are capable of growth at some of the highest temperatures and extremes of pH of all known organisms. Phylogenetic rRNA-encoding DNA analysis places many of the hyperthermophilic Archaea (species with an optimum growth > or = 80 degrees C) at the base of the universal tree of life, suggesting that thermophiles were among the first forms of life on earth. Very few viruses have been identified from Archaea as compared to Bacteria and Eukarya. We report here the structure of a hyperthermophilic virus isolated from an archaeal host found in hot springs in Yellowstone National Park. The sequence of the circular double-stranded DNA viral genome shows that it shares little similarity to other known genes in viruses or other organisms. By comparing the tertiary and quaternary structures of the coat protein of this virus with those of a bacterial and an animal virus, we find conformational relationships among all three, suggesting that some viruses may have a common ancestor that precedes the division into three domains of life >3 billion years ago.

Morphology and genome organization of the virus PSV of the hyperthermophilic archaeal genera Pyrobaculum and Thermoproteus: a novel virus family, the Globuloviridae

A novel virus, termed Pyrobaculum spherical virus (PSV), is described that infects anaerobic hyperthermophilic archaea of the genera Pyrobaculum and Thermoproteus. Spherical enveloped virions, about 100 nm in diameter, contain a major multimeric 33-kDa protein and host-derived lipids. A viral envelope encases a superhelical nucleoprotein core containing linear double-stranded DNA. The PSV infection cycle does not cause lysis of host cells. The viral genome was sequenced and contains 28 337 bp. The genome is unique for known archaeal viruses in that none of the genes, including that encoding the major structural protein, show any significant sequence matches to genes in public sequence databases. Exceptionally for an archaeal double-stranded DNA virus, almost all the recognizable genes are located on one DNA strand. The ends of the genome consist of 190-bp inverted repeats that contain multiple copies of short direct repeats. The two DNA strands are probably covalently linked at their termini. On the basis of the unusual morphological and genomic properties of this DNA virus, we propose to assign PSV to a new viral family, the Globuloviridae.

AFV1, a novel virus infecting hyperthermophilic archaea of the genus acidianus

We describe a novel virus, AFV1, of the hyperthermophilic archaeal genus Acidianus. Filamentous virions are covered with a lipid envelope and contain at least five different proteins with molecular masses in the range of 23–130 kDa and a 20.8-kb-long linear double-stranded DNA. The virus has been assigned to the family Lipothrixviridae on the basis of morphotypic characteristics. Host range is confined to several strains of Acidianus and the virus persists in its hosts in a stable carrier state. The latent period of virus infection is about 4 h. Viral DNA was sequenced and sequence similarities were found to the lipothrixvirus SIFV, the rudiviruses SIRV1 and SIRV2, as well as to conjugative plasmids and chromosomes of the genus Sulfolobus. Exceptionally for the linear genomes of archaeal viruses, many short direct repeats, with the sequence TTGTT or close variants thereof, are closely clustered over 300 bp at each end of the genome. They are reminiscent of the telomeric ends of linear eukaryal chromosomes.

Holliday junction resolving enzymes of archaeal viruses SIRV1 and SIRV2

In the final stages of genetic recombination, Holliday junction resolving enzymes transform the four-way DNA intermediate into two duplex DNA molecules by introducing pairs of staggered nicks flanking the junction. This fundamental process is apparently common to cells from all three domains of life. Two cellular resolving enzymes from extremely thermophilic representatives of both kingdoms of the domain Archaea, the euryarchaeon Pyrococcus furiosus and the crenarchaeon Sulfolobus solfataricus, have been described recently. Here we report for the first time the isolation, purification and characterization of Holliday junction cleaving enzymes (Hjc) from two archaeal viruses. Both viruses, SIRV1 and SIRV2, infect Sulfolobus islandicus. Their Hjcs both consist of 121 amino acid residues (aa) differing only by 18 aa. Both proteins bind selectively to synthetic Holliday-structure analogues with an apparent dissociation constant of 25 nM. In the presence of Mg 2+ the enzymes produce identical cleavage patterns near the junction. While S. islandicus shows optimal growth at about 80°C, the nucleolytic activities of recombinant SIRV2 Hjc was highest between 45°C and 70°C. Based on their specificity for four-way DNA structures the enzymes may play a general role in genetic recombination, DNA repair and the resolution of replicative intermediates.