Identification of two origins of replication in the single chromosome of the archaeon Sulfolobus solfataricus

Eukaryotic chromosomes possess multiple origins of replication, whereas bacterial chromosomes are replicated from a single origin. The archaeon Pyrococcus abyssi also appears to have a single origin, suggesting a common rule for prokaryotes. However, in the current work, we describe the identification of two active origins of replication in the single chromosome of the hyperthermophilic archaeon Sulfolobus solfataricus. Further, we identify conserved sequence motifs within the origins that are recognized by a family of three Sulfolobus proteins that are homologous to the eukaryotic initiator proteins Orc1 and Cdc6. We demonstrate that the two origins are recognized by distinct subsets of these Orc1/Cdc6 homologs. These data, in conjunction with an analysis of the levels of the three Orc1/Cdc6 proteins in different growth phases and cell cycle stages, lead us to propose a model for the roles for these proteins in modulating origin activity.     

Identification of replication origins in archaeal genomes based on the Z-curve method

The Z-curve is a three-dimensional curve that constitutes a unique representation of a DNA sequence, i.e., both the Z-curve and the given DNA sequence can be uniquely reconstructed from the other. We employed Z-curve analysis to identify one replication origin in the Methanocaldococcus jannaschii genome, two replication origins in the Halobacterium species NRC-1 genome and one replication origin in the Methanosarcina mazei genome. One of the predicted replication origins of Halobacterium species NRC-1 is the same as a replication origin later identified by in vivo experiments. The Z-curve analysis of the Sulfolobus solfataricus P2 genome suggested the existence of three replication origins, which is also consistent with later experimental results. This review aims to summarize applications of the Z-curve in identifying replication origins of archaeal genomes, and to provide clues about the locations of as yet unidentified replication origins of the Aeropyrum pernix K1, Methanococcus maripaludis S2, Picrophilus torridus DSM 9790 and Pyrobaculum aerophilum str. IM2 genomes.     

Multiple replication origins of the archaeon Halobacterium species NRC-1

The genomic sequence of the halophilic archaeon Halobacterium NRC-1 has been analyzed by the Z curve method. The Z curve is a three-dimensional curve that uniquely represents a given DNA sequence. Based on the known behaviors of the Z curves for the archaea whose replication origins have been identified, the analysis of the Z curve for the genome of Halobacterium NRC-1 strongly suggests that the large genome has two replication origins, oriC1 (921,863-922,014) and oriC2 (1,806,444-1,807,229), which are located at two sharp peaks of the Z curve. These two regions are next to the cdc6 genes and contain multiple copies of stretches of G and C, i.e., ggggtgggg and ccccacccc, which may also be regarded as direct and inverted repeats. Based on the above analysis, a model of replication of Halobacterium NRC-1 with two replication origins and two termini has been proposed. The experimental confirmation of this model would constitute the first example of multiple replication origins of archaea, which will finally provide much insight into the understanding of replication mechanisms of eukaryotic organisms, including human. In addition, the potential multiple replication origins of the archaeon Sulfolobus solfataricus are suggested by the analysis based on the Z curve method.     

Molecular determinants of origin discrimination by Orc1 initiators in archaea

Unlike bacteria, many eukaryotes initiate DNA replication from genomic sites that lack apparent sequence conservation. These loci are identified and bound by the origin recognition complex (ORC), and subsequently activated by a cascade of events that includes recruitment of an additional factor, Cdc6. Archaeal organisms generally possess one or more Orc1/Cdc6 homologs, belonging to the Initiator clade of ATPases associated with various cellular activities (AAA(+)) superfamily; however, these proteins recognize specific sequences within replication origins. Atomic resolution studies have shown that archaeal Orc1 proteins contact double-stranded DNA through an N-terminal AAA(+) domain and a C-terminal winged-helix domain (WHD), but use remarkably few base-specific contacts. To investigate the biochemical effects of these associations, we mutated the DNA-interacting elements of the Orc1-1 and Orc1-3 paralogs from the archaeon Sulfolobus solfataricus, and tested their effect on origin binding and deformation. We find that the AAA(+) domain has an unpredicted role in controlling the sequence selectivity of DNA binding, despite an absence of base-specific contacts to this region. Our results show that both the WHD and ATPase region influence origin recognition by Orc1/Cdc6, and suggest that not only DNA sequence, but also local DNA structure help define archaeal initiator binding sites.     

Three eukaryote-like Orc1/Cdc6 proteins functionally interact and mutually regulate their activities of binding to the replication origin in the hyperthermophilic archaeon Sulfolobus solfataricus P2

The crenarchaeon Sulfolobus solfataricus has the potential to be a powerful model system to understand the central mechanism of eukaryotic DNA replication because it contains three active origins of replication and three eukaryote-like Orc1/Cdc6 proteins. However, it is not known whether these SsoCdc6 proteins can functionally interact and collectively contribute to DNA replication initiation. In the current work, we found that SsoCdc6-1 stimulates DNA-binding activities of SsoCdc6-3. In contrast, SsoCdc6-3 inhibits those of both SsoCdc6-1 and SsoCdc6-2. These regulatory functions are differentially affected by the C-terminal domains of these SsoCdc6 proteins. These data, in conjunction with studies on physical interactions between these replication initiators by bacterial two-hybrid and pull-down/Western blot assays, lead us to propose the possibility that multiple SsoCdc6 proteins might coordinately regulate DNA replication in the archaeon species. This is the first report on the functional interaction among the archaeal multiple Cdc6 proteins to regulate DNA replication.     

The sub-cellular localization of Sulfolobus DNA replication

Analyses of the DNA replication-associated proteins of hyperthermophilic archaea have yielded considerable insight into the structure and biochemical function of these evolutionarily conserved factors. However, little is known about the regulation and progression of DNA replication in the context of archaeal cells. In the current work, we describe the generation of strains of Sulfolobus solfataricus and Sulfolobus acidocaldarius that allow the incorporation of nucleoside analogues during DNA replication. We employ this technology, in conjunction with immunolocalization analyses of replisomes, to investigate the sub-cellular localization of nascent DNA and replisomes. Our data reveal a peripheral localization of replisomes in the cell. Furthermore, while the two replication forks emerging from any one of the three replication origins in the Sulfolobus chromosome remain in close proximity, the three origin loci are separated.     

The chromosome replication machinery of the archaeon Sulfolobus solfataricus

In the three domains of life, the archaea, bacteria, and eukarya, there are two general lineages of DNA replication proteins: the bacterial and the eukaryal/archaeal lineages. The hyperthermophilic archaeon Sulfolobus solfataricus provides an attractive model for biochemical study of DNA replication. Its relative simplicity in both genomic and biochemical contexts, together with high protein thermostability, has already provided insight into the function of the more complex yet homologous molecules of the eukaryotic domain. Here, we provide an overview of recent insights into the functioning of the chromosome replication machinery of S. solfataricus, focusing on some of the relatively well characterized core components that act at the DNA replication fork.     

Archaeal cell cycle progress

The discovery of multiple chromosome replication origins in Sulfolobus species has added yet another eukaryotic trait to the archaea, and brought new levels of complexity to the cell cycle in terms of initiation of chromosome replication, replication termination and chromosome decatenation. Conserved repeated DNA elements--origin recognition boxes--have been identified in the origins of replication, and shown to bind the Orc1/Cdc6 proteins involved in cell cycle control. The origin recognition boxes aid in the identification and characterization of new origins, and their conservation suggests that most archaea have a similar replication initiation mechanism. Cell-cycle-dependent variation in Orc1/Cdc6 levels has been demonstrated, reminiscent of variations in cyclin levels during the eukaryotic cell cycle. Information about archaeal chromosome segregation is also accumulating, including the identification of a protein that binds to short regularly spaced repeats that might constitute centromere-like elements. In addition, studies of cell-cycle-specific gene expression have potential to reveal, in the near future, missing components in crenarchaeal chromosome replication, genome segregation and cell division. Together with an increased number of physiological and cytological investigations of the overall organization of the cell cycle, rapid progress of the archaeal cell cycle field is evident, and archaea, in particular Sulfolobus species, are emerging as simple and powerful models for the eukaryotic cell cycle.     

Regulation of the functional interactions between archaeal eukaryote-like Cdc6/Orc1 proteins on the replication origin by two different mechanisms

The crenarchaeon Sulfolobus solfataricus contains three active origins of replication and three eukaryote-like Cdc6/Orc1 proteins known as SsoCdc6 proteins. It has the potential to become a powerful model system in understanding the central mechanism of the eukaryotic DNA replication. In this research, we designed a group of duplex DNA substrates containing specific origin recognition boxes (ORBs) of the archaeon and identified the DNA-binding activities of different SsoCdc6 proteins. Furthermore, we showed that the DNA-protein interaction between the DNA substrate and the SsoCdc6-1 or SsoCdc6-3 strikingly regulated their DNA-binding activities of each other on the origin. On the other hand, the protein-protein interactions between SsoCdc6-1 and SsoCdc6-2 were observed to mutually modulate the stimulating or inhibitive effects on the DNA-binding activities of each other. Thus, two different mechanisms were demonstrated to be involved in the regulations of the functions of the SsoCdc6 proteins on the replication origins. The results of this study imply that the interactions between multiple SsoCdc6 proteins and origin DNA collectively contribute to the positive or negative regulation of DNA replication initiation in the archaeon species.     

Divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on modulating the loading of the MCM helicase onto the origins of the hyperthermophilic archaeon Sulfolobus solfataricus P2

The Cdc6 protein has been suggested as a loader for the eukaryotic MCM helicase. Archaeal replication machinery represents a core version of that in eukaryotes. In the current work, three eukaryotic Orc1/Cdc6 homologs (SsoCdc6-1, -2, and -3) from crenarchaeon Sulfolobus solfataricus were shown to have totally different effects on the interactions with SsoMCM helicase. SsoCdc6-2 stimulates the binding of the SsoMCM onto the origin DNA, but SsoCdc6-1 and SsoCdc6-3 significantly inhibit the loading activities, and these inhibitive effects can not be reversed by the stimulation of SsoCdc6-2. Using pull-down assays, we showed that three SsoCdc6 proteins interacted physically with the SsoMCM. Furthermore, the C-terminal domains of SsoCdc6 proteins were shown to physically and functionally affect the interactions with SsoMCM. This is the first report on the divergent functions of multiple eukaryote-like Orc1/Cdc6 proteins on regulating the loading of the MCM helicase onto the origins in the archaeon.     

Strand annealing and terminal transferase activities of a B-family DNA polymerase

DNA replication polymerases have the inherent ability to faithfully and rapidly copy a DNA template according to precise Watson-Crick base pairing. The primary B-family DNA replication polymerase (Dpo1) in the hyperthermophilic archaeon, Sulfolobus solfataricus, is shown here to possess a remarkable DNA stabilizing ability for maintaining weak base pairing interactions to facilitate primer extension. This thermal stabilization by Dpo1 allowed for template-directed synthesis at temperatures more than 30 °C above the melting temperature of naked DNA. Surprisingly, Dpo1 also displays a competing terminal deoxynucleotide transferase (TdT) activity unlike any other B-family DNA polymerase. Dpo1 is shown to elongate single-stranded DNA in template-dependent and template-independent manners. Experiments with different homopolymeric templates indicate that initial deoxyribonucleotide incorporation is complementary to the template. Rate-limiting steps that include looping back and annealing to the template allow for a unique template-dependent terminal transferase activity. The multiple activities of this unique B-family DNA polymerase make this enzyme an essential component for DNA replication and DNA repair for the maintenance of the archaeal genome at high temperatures.     

A new phosphotriesterase from Sulfolobus acidocaldarius and its comparison with the homologue from Sulfolobus solfataricus

The phosphotriesterase PTE, identified in the soil bacterium Pseudomonas diminuta, is thought to have evolved in the last several decades to degrade the pesticide paraoxon with proficiency approaching the limit of substrate diffusion (k(cat)/K(M) of 4 x 10(7)M(-1)s(-1)). It belongs to the amidohydrolase superfamily, but its evolutionary origin remains obscure. The enzyme has important potentiality in the field of the organophosphate decontamination. Recently we reported on the characterization of an archaeal member of the amidohydrolase superfamily, namely Sulfolobus solfataricus, showing low but significant and extremely thermostable paraoxonase activity (k(cat)/K(M) of 4 x 10(3)M(-1)s(-1)). Looking for other thermostable phosphotriesterases we assayed, among others, crude extracts of Sulfolobus acidocaldarius and detected activity. Since the genome of S. acidocaldarius has been recently reported, we identified there an open reading frame highly related to the S. solfataricus enzyme. The gene was cloned, the protein overexpressed in Escherichia coli, purified, and proven to have paraoxonase activity. A comparative analysis detected some significant differences between the two archaeal enzymes.     

3-hydroxy-3-methylglutaryl coenzyme A reductase of Sulfolobus solfataricus: DNA sequence, phylogeny, expression in Escherichia coli of the hmgA gene, and purification and kinetic characterization of the gene product.

The gene (hmgA) for 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase (EC 1.1.1.34) from the thermophilic archaeon Sulfolobus solfataricus P2 was cloned and sequenced. S. solfataricus HMG-CoA reductase exhibited a high degree of sequence identity (47%) to the HMG-CoA reductase of the halophilic archaeon Haloferax volcanii. Phylogenetic analyses of HMG-CoA reductase protein sequences suggested that the two archaeal genes are distant homologs of eukaryotic genes. The only known bacterial HMG-CoA reductase, a strictly biodegradative enzyme from Pseudomonas mevalonii, is highly diverged from archaeal and eukaryotic HMG-CoA reductases. The S. solfataricus hmgA gene encodes a true biosynthetic HMG-CoA reductase. Expression of hmgA in Escherichia coli generated a protein that both converted HMG-CoA to mevalonate and cross-reacted with antibodies raised against rat liver HMG-CoA reductase. S. solfataricus HMG-CoA reductase was purified in 40% yield to a specific activity of 17.5 microU per mg at 50 degrees C by a sequence of steps that included heat treatment, ion-exchange chromatography, hydrophobic interaction chromatography, and affinity chromatography. The final product was homogeneous, as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The substrate was (S)- not (R)-HMG-CoA; the reductant was NADPH not NADH. The Km values for HMG-CoA (17 microM) and NADPH (23 microM) were similar in magnitude to those of other biosynthetic HMG-CoA reductases. Unlike other HMG-CoA reductases, the enzyme was stable at 90 degrees C and was optimally active at pH 5.5 and 85 degrees C.     

A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases.

The gene encoding the thermostable DNA polymerase from the archaeon Sulfolobus solfataricus (strain MT 4) was isolated by means of two degenerate oligonucleotide probes. They were designed on the basis of partial enzyme amino acid sequences. The gene was found to encode a 882 residues polypeptide chain with a deduced molecular mass of about 100 kDa. By comparison with other archaeal genes, putative regulatory sites were identified in the gene-flanking regions. By computer-assisted homology search, several sequence similarities among S. solfataricus and family B DNA polymerases were found. In addition, conserved sequence motifs, implicated in the 3'-5' exonuclease activity of E. coli DNA polymerase I and shared by various family A and B DNA polymerases, were also identified. This result suggests that the proofreading domains of all these enzymes are evolutionarily related.     

Recurrent paralogy in the evolution of archaeal chaperonins.

Chaperonins are multisubunit double-ring complexes that mediate the folding of nascent proteins [1] [2]. In bacteria, chaperonins are homo-oligomeric and are composed of seven-membered rings. Eukaryotic and most archaeal chaperonin rings are eight-membered and exhibit varying degrees of hetero-oligomerism [3] [4]. We have cloned and sequenced seven new genes encoding chaperonin subunits from the crenarchaeotes Sulfolobus solfataricus, S. acidocaldarius, S. shibatae and Desulfurococcus mobilis. Although some archaeal genomes possess a single chaperonin gene, most have two. We describe a third chaperonin-encoding gene (TF55-gamma) from two Sulfolobus species; phylogenetic analyses indicate that the gene duplication producing TF55-gamma occurred within crenarchaeal evolution. The presence of TF55-gamma in Sulfolobus correlates with their unique nine-membered chaperonin rings. Duplicate genes (paralogs) for chaperonins within archaeal genomes very often resemble each other more than they resemble chaperonin genes from other archaea. Our phylogenetic analyses suggest multiple independent gene duplications - at least seven among the archaea examined. The persistence of paralogous genes for chaperonin subunits in multiple archaeal lineages may involve a process of co-evolution, where chaperonin subunit heterogeneity changes independently of selection on function.     

Genetic and physical mapping of DNA replication origins in Haloferax volcanii

The halophilic archaeon Haloferax volcanii has a multireplicon genome, consisting of a main chromosome, three secondary chromosomes, and a plasmid. Genes for the initiator protein Cdc6/Orc1, which are commonly located adjacent to archaeal origins of DNA replication, are found on all replicons except plasmid pHV2. However, prediction of DNA replication origins in H. volcanii is complicated by the fact that this species has no less than 14 cdc6/orc1 genes. We have used a combination of genetic, biochemical, and bioinformatic approaches to map DNA replication origins in H. volcanii. Five autonomously replicating sequences were found adjacent to cdc6/orc1 genes and replication initiation point mapping was used to confirm that these sequences function as bidirectional DNA replication origins in vivo. Pulsed field gel analyses revealed that cdc6/orc1-associated replication origins are distributed not only on the main chromosome (2.9 Mb) but also on pHV1 (86 kb), pHV3 (442 kb), and pHV4 (690 kb) replicons. Gene inactivation studies indicate that linkage of the initiator gene to the origin is not required for replication initiation, and genetic tests with autonomously replicating plasmids suggest that the origin located on pHV1 and pHV4 may be dominant to the principal chromosomal origin. The replication origins we have identified appear to show a functional hierarchy or differential usage, which might reflect the different replication requirements of their respective chromosomes. We propose that duplication of H. volcanii replication origins was a prerequisite for the multireplicon structure of this genome, and that this might provide a means for chromosome-specific replication control under certain growth conditions. Our observations also suggest that H. volcanii is an ideal organism for studying how replication of four replicons is regulated in the context of the archaeal cell cycle.     

Sulfolobus solfataricus translation initiation factor 1 stimulates translation initiation complex formation

The eukaryotic translation initiation factor 1 binds to the ribosome during translation initiation. It is instrumental for initiator-tRNA and mRNA binding, and has a function in selection of the authentic start codon. Here, we show that the archaeal homolog aIF1 has analogous functions. The aIF1 protein of the archaeon Sulfolobus solfataricus is bound to the small ribosomal subunit during translation initiation and accelerates binding of initiator-tRNA and mRNA to the ribosome. Accordingly, aIF1 stimulated translation of an mRNA in a S. solfataricus in vitro translation system. Moreover, this study suggested that the C terminus of the factor is of relevance for its function.     

Four chromosome replication origins in the archaeon Pyrobaculum calidifontis

Replication origins were mapped in hyperthermophilic crenarchaea, using high‐throughput sequencing‐based marker frequency analysis. We confirm previous origin mapping in Sulfolobus acidocaldarius, and demonstrate that the single chromosome of Pyrobaculum calidifontis contains four replication origins, the highest number detected in a prokaryotic organism. The relative positions of the origins in both organisms coincided with regions enriched in highly conserved (core) archaeal genes. We show that core gene distribution provides a useful tool for origin identification in archaea, and predict multiple replication origins in a range of species. One of the P. calidifontis origins was mapped in detail, and electrophoretic mobility shift assays demonstrated binding of the Cdc6/Orc1 replication initiator protein to a repeated sequence element, denoted Orb‐1, within the origin. The high‐throughput sequencing approach also allowed for an annotation update of both genomes, resulting in the restoration of open reading frames encoding proteins involved in, e.g., sugar, nitrate and energy metabolism, as well as in glycosylation and DNA repair.     

Sister chromatid junctions in the hyperthermophilic archaeon Sulfolobus solfataricus

Although the Archaea exhibit an intriguing combination of bacterial- and eukaryotic-like features, it is not known how these prokaryotic cells segregate their chromosomes before the process of cell division. In the course of our analysis of the third replication origin in the archaeon Sulfolobus solfataricus, we identify and characterise sister chromatid junctions in this prokaryote. This pairing appears to be mediated by hemicatenane-like structures, and we provide evidence that these junctions persist in both replicating and postreplicative cells. These data, in conjunction with fluorescent in situ hybridisation analyses, suggest that Sulfolobus chromosomes have a significant period of postreplicative sister chromatid synapsis, a situation that is more reminiscent of eukaryotic than bacterial chromosome segregation mechanisms.