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.     

Insight into initiator-DNA interactions: a lesson from the archaeal ORC

Although initiation of DNA replication is considered to be highly coordinated through multiple protein-DNA and protein-protein interactions, it is poorly understood how particular locations within the eukaryotic chromosome are selected as origins of DNA replication. Here, we discuss recent reports that present structural information on the interaction characteristics of the archaeal orthologues of the eukaryotic origin recognition complex with their cognate binding sequences. Since the archaeal replication system is postulated as a simplified version of the one in eukaryotes, by analogy, these works provide insights into the functions of the eukaryotic initiator proteins.     

The archaeal cell cycle: current issues

The recently discovered structural similarities between the archaeal Orc1/Cdc6 and bacterial DnaA initiator proteins for chromosome replication have exciting implications for cell cycle regulation. Together with current attempts to identify archaeal chromosome replication origins, the information is likely to yield fundamental insights into replication control in both archaea and eukaryotes within the near future. Several proteins that affect, or are likely to affect, chromatin structure and genome segregation in archaea have been described recently, including Sph1 and 2, ScpA and B, Sir2, Alba and Rio1p. Important insights into the properties of the MinD and FtsZ cell division proteins, and of putative cytoskeletal elements, have recently been gained in bacteria. As these proteins also are present among archaea, it is likely that the new information will also be essential for understanding archaeal genome segregation and cell division. A series of interesting cell cycle issues has been brought to light through the discovery of the novel Nanoarchaeota phylum, and these are outlined briefly. Exciting areas for extended cell cycle investigations of archaea are identified, including termination of chromosome replication, application of in situ cytological techniques for localization of cell cycle proteins and the regulatory roles of GTP-binding proteins and small RNAs.     

Protein-protein interactions in the archaeal core replisome

Most of the core components of the archaeal chromosomal DNA replication apparatus share significant protein sequence similarity with eukaryotic replication factors, making the Archaea an excellent model system for understanding the biology of chromosome replication in eukaryotes. The present review summarizes current knowledge of how the core components of the archaeal chromosome replication apparatus interact with one another to perform their essential functions.     

Stabilization of an archaeal DNA-sliding clamp protein, PCNA, by proteasome-activating nucleotidase gene knockout in Haloferax volcanii

Many details of structure, function and substrate specificity of eukaryotic proteasomal systems have been elucidated. This information far-exceeds that available for the archaeal and bacterial counterparts. While structural and functional studies have provided some insight into the workings of prokaryotic proteasomes, the question of substrate targeting and global cellular influence remain largely unaddressed. In this communication, we report an over 720-fold increase in the half-life of the DNA-sliding clamp protein proliferating cell nuclear antigen after knockout of the panA gene, encoding a proteasome-activating nucleotidase A, on the chromosome of the halophilic archaeon Haloferax volcanii . This discovery marks the first identification of a protein stabilized by an archaeal proteasome mutation and provides a starting point for investigations into substrate recognition mechanisms. The findings also begin to address the functional role of proteasomal systems within the scope of the archaeal cell.     

Identification of short 'eukaryotic' Okazaki fragments synthesized from a prokaryotic replication origin

Although archaeal genomes encode proteins similar to eukaryotic replication factors, the hyperthermophilic archaeon Pyrococcus abyssi replicates its circular chromosome at a high rate from a single origin (oriC) as in Bacteria. In further elucidating the mechanism of archaeal DNA replication, we have studied the elongation step of DNA replication in vivo. We have detected, in two main archaeal phyla, short RNA-primed replication intermediates whose structure and length are very similar to those of eukaryotic Okazaki fragments. Mapping of replication initiation points further showed that discontinuous DNA replication in P. abyssi starts at a well-defined site within the oriC recently identified in this hyperthermophile. Short Okazaki fragments and a high replication speed imply a very efficient turnover of Okazaki fragments in Archaea. Archaea therefore have a unique replication system showing mechanistic similarities to both Bacteria and Eukarya.     

Chromosome replication, nucleoid segregation and cell division in archaea

Recent progress in cell cycle analysis of archaea has included the identification of putative chromosome replication origins, novel DNA polymerases and an unusual mode of cell cycle organization featuring multiple copies of the chromosome and asymmetric cell divisions. Genome sequence data indicate that in crenarchaea, the 'ubiquitous' FtsZ/MinD-based prokaryotic cell division apparatus is absent and division therefore must occur by unique, as-yet-unidentified mechanisms. The evolutionary and functional relationships between the archaeal Cdc6 protein and bacterial and eukaryal replication initiation factors are discussed.     

The structure of bacterial DnaA: implications for general mechanisms underlying DNA replication initiation

The initiation of DNA replication is a key event in the cell cycle of all organisms. In bacteria, replication initiation occurs at specific origin sequences that are recognized and processed by an oligomeric complex of the initiator protein DnaA. We have determined the structure of the conserved core of the Aquifex aeolicus DnaA protein to 2.7 A resolution. The protein comprises an AAA+ nucleotide-binding fold linked through a long, helical connector to an all-helical DNA-binding domain. The structure serves as a template for understanding the physical consequences of a variety of DnaA mutations, and conserved motifs in the protein suggest how two critical aspects of origin processing, DNA binding and homo-oligomerization, are mediated. The spatial arrangement of these motifs in DnaA is similar to that of the eukaryotic-like archaeal replication initiation factor Cdc6/Orc1, demonstrating that mechanistic elements of origin processing may be conserved across bacterial, archaeal and eukaryotic domains of life.     

The promiscuous primase

DNA primases are essential for the initiation of DNA replication and progression of the replication fork. Recent phylogenetic analyses coupled with biochemical and structural studies have revealed that the arrangement of catalytic residues within the archaeal and eukaryotic primase has significant similarity to those of the Pol X family of DNA-repair polymerases. Furthermore, two additional groups of enzymes, the ligase/primase of the bacterial nonhomologous end-joining machinery and a putative replicase from an archaeal plasmid have shown striking functional and structural similarities to the core primase. The promiscuous nature of the archaeal primases suggests that these proteins might have additional roles in DNA repair in the archaea.     

Archaeal protein translocation crossing membranes in the third domain of life.

Proper cell function relies on correct protein localization. As a first step in the delivery of extracytoplasmic proteins to their ultimate destinations, the hydrophobic barrier presented by lipid-based membranes must be overcome. In contrast to the well-defined bacterial and eukaryotic protein translocation systems, little is known about how proteins cross the membranes of archaea, the third and most recently described domain of life. In bacteria and eukaryotes, protein translocation occurs at proteinaceous sites comprised of evolutionarily conserved core components acting in concert with other, domain-specific elements. Examination of available archaeal genomes as well as cloning of individual genes from other archaeal strains reveals the presence of homologues to selected elements of the bacterial or eukaryotic translocation machines. Archaeal genomic searches, however, also reveal an apparent absence of other, important components of these two systems. Archaeal translocation may therefore represent a hybrid of the bacterial and eukaryotic models yet may also rely on components or themes particular to this domain of life. Indeed, considering the unique chemical composition of the archaeal membrane as well as the extreme conditions in which archaea thrive, the involvement of archaeal-specific translocation elements could be expected. Thus, understanding archaeal protein translocation could reveal the universal nature of certain features of protein translocation which, in some cases, may not be readily obvious from current comparisons of bacterial and eukaryotic systems. Alternatively, elucidation of archaeal translocation could uncover facets of the translocation process either not yet identified in bacteria or eukaryotes, or which are unique to archaea. In the following, the current status of our understanding of protein translocation in archaea is reviewed.     

Identifying bacterial and archaeal homologs of pentameric ligand-gated ion channel (pLGIC) family using domain-based and alignment-based approaches

Identification of bacterial and archaeal counterparts to eukaryotic ion channels has greatly facilitated studies of structural biophysics of the channels. Often, searches based only on sequence alignment tools are inadequate for discovering such distant bacterial and archaeal counterparts. We address the discovery of bacterial and archaeal members of the Pentameric Ligand-Gated Ion Channel (pLGIC) family by a combination of four computational methods. One domain-based method involves retrieval of proteins with pLGIC-relevant domains by matching those domains to previously established domain templates in the InterPro family of databases. The second domain-based method involves searches using ungapped de-novo motifs discovered by MEME which were trained with well characterized members of the pLGIC family. The third and fourth methods involve the use of two sequence alignment search algorithms BLASTp and psiBLAST respectively. The sequences returned from all methods were screened by having the correct topology for pLGIC's, and by returning an annotated member of this family as one of the first ten hits using BLASTp against a comprehensive database of eukaryotic proteins. We found the domain based searches to have high specificity but low sensitivity, while the sequence alignment methods have higher sensitivity but lower specificity. The four methods together discovered 69 putative bacterial and archaeal members of the pLGIC family. We ranked and divide the 69 proteins into groups according to the similarity of their domain compositions with known eukaryotic pLGIC's. One especially notable group is more closely related to eukaryotic pLGIC's than to any other known protein family, and has the overall topology of pLGIC's, but the functional domains they contain are sufficiently different from those found in known pLGIC's that they do not score very well against the pLGIC domain templates. We conclude that multiple methods used in a coordinated fashion outperform any single method for identifying likely distant bacterial and archaeal proteins that may provide useful models for important eukaryotic channel function. We note also that the methods used here are largely standard and readily accessible. The novelty is in the effectiveness of a strategy that combines these methods for identifying bacterial and archea relatives of this family. Therefore the paper may serve as a template for a broad group of workers to reliably identify bacterial and archaeal counterparts to eukaryotic proteins.     

Archaeal DNA replication: spotlight on a rapidly moving field

The replication of DNA is a fundamental step in the cell cycle, which must be coordinated with cell division to ensure that the daughter cells inherit the same genomic material as the parental cell. The recently published complete genome sequences of some archaeal species together with preliminary biochemical studies suggest that the Archaea quite likely duplicate their chromosome by using replication machinery that seems to be a simplified version of the eukaryotic machinery, although their metabolic facets and their cellular morphology are prokaryotic-like. This review is focused on recent progress on the structural and functional analysis of proteins and enzymes involved in the initiation and elongation steps of DNA replication in Archaea. Differences between the genome replication apparatus of the Euryarchaea and the Crenarchaea (the two main phylogenetic divisions of the Archaea domain) are highlighted.     

The archaeo-eukaryotic GINS proteins and the archaeal primase catalytic subunit PriS share a common domain

Primase and GINS are essential factors for chromosomal DNA replication in eukaryotic and archaeal cells. Here we describe a previously undetected relationship between the C-terminal domain of the catalytic subunit (PriS) of archaeal primase and the B-domains of the archaeo-eukaryotic GINS proteins in the form of a conserved structural domain comprising a three-stranded antiparallel β-sheet adjacent to an α-helix and a two-stranded β-sheet or hairpin. The presence of a shared domain in archaeal PriS and GINS proteins, the genes for which are often found adjacent on the chromosome, suggests simple mechanisms for the evolution of these proteins.     

The structure and function of the bacterial chromosome

Advances in microscopic and cell biological techniques have considerably improved our understanding of bacterial chromosome organization and dynamics. The nucleoid was formerly perceived to be an amorphous entity divided into ill-defined domains of supercoiling that are randomly deposited in the cell. Recent work, however, has demonstrated a remarkable degree of spatial organization. A highly ordered chromosome structure, established while DNA replication and partitioning are in progress, is maintained and propagated during growth. Duplication of the chromosome and partitioning of the newly generated daughter strands are interwoven processes driven by the dynamic interplay between the synthesis, segregation and condensation of DNA. These events are intimately coupled with the bacterial cell cycle and exhibit a previously unanticipated complexity reminiscent of eukaryotic systems.     

New genes with old modus operandi. The connection between supercoiling and partitioning of DNA in Escherichia coli

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as ‘red herrings’ in the search for ‘true’ partition functions, are again recognized as being important for chromosome partitioning in E. coli.     

Identifying bacterial and archaeal homologs of pentameric ligand-gated ion channel (pLGIC) family using domain-based and alignment-based approaches

Identification of bacterial and archaeal counterparts to eukaryotic ion channels has greatly facilitated studies of structural biophysics of the channels. Often, searches based only on sequence alignment tools are inadequate for discovering such distant bacterial and archaeal counterparts. We address the discovery of bacterial and archaeal members of the Pentameric Ligand-Gated Ion Channel (pLGIC) family by a combination of four computational methods. One domain-based method involves retrieval of proteins with pLGIC-relevant domains by matching those domains to previously established domain templates in the InterPro family of databases. The second domain-based method involves searches using ungapped de-novo motifs discovered by MEME which were trained with well characterized members of the pLGIC family. The third and fourth methods involve the use of two sequence alignment search algorithms BLASTp and psiBLAST respectively. The sequences returned from all methods were screened by having the correct topology for pLGIC's, and by returning an annotated member of this family as one of the first ten hits using BLASTp against a comprehensive database of eukaryotic proteins. We found the domain based searches to have high specificity but low sensitivity, while the sequence alignment methods have higher sensitivity but lower specificity. The four methods together discovered 69 putative bacterial and archaeal members of the pLGIC family. We ranked and divide the 69 proteins into groups according to the similarity of their domain compositions with known eukaryotic pLGIC's. One especially notable group is more closely related to eukaryotic pLGIC's than to any other known protein family, and has the overall topology of pLGIC's, but the functional domains they contain are sufficiently different from those found in known pLGIC's that they do not score very well against the pLGIC domain templates. We conclude that multiple methods used in a coordinated fashion outperform any single method for identifying likely distant bacterial and archaeal proteins that may provide useful models for important eukaryotic channel function. We note also that the methods used here are largely standard and readily accessible. The novelty is in the effectiveness of a strategy that combines these methods for identifying bacterial and archea relatives of this family. Therefore the paper may serve as a template for a broad group of workers to reliably identify bacterial and archaeal counterparts to eukaryotic proteins.     

The bacterial nucleoid: a highly organized and dynamic structure

Recent advances in bacterial cell biology have revealed unanticipated structural and functional complexity, reminiscent of eukaryotic cells. Particular progress has been made in understanding the structure, replication, and segregation of the bacterial chromosome. It emerged that multiple mechanisms cooperate to establish a dynamic assembly of supercoiled domains, which are stacked in consecutive order to adopt a defined higher-level organization. The position of genetic loci on the chromosome is thereby linearly correlated with their position in the cell. SMC complexes and histone-like proteins continuously remodel the nucleoid to reconcile chromatin compaction with DNA replication and gene regulation. Moreover, active transport processes ensure the efficient segregation of sister chromosomes and the faithful restoration of nucleoid organization while DNA replication and condensation are in progress.     

GINS, a central nexus in the archaeal DNA replication fork

In eukaryotes, the GINS complex is essential for DNA replication and has been implicated as having a role at the replication fork. This complex consists of four paralogous GINS subunits, Psf1, Psf2, Psf3 and Sld5. Here, we identify an archaeal GINS homologue as a direct interaction partner of the MCM helicase. The core archaeal GINS complex contains two subunits that are poorly conserved homologues of the eukaryotic GINS subunits, in complex with a protein containing a domain homologous to the DNA-binding domain of bacterial RecJ. Interaction studies show that archaeal GINS interacts directly with the heterodimeric core primase. Our data suggest that GINS is important in coordinating the architecture of the replication fork and provide a mechanism to couple progression of the MCM helicase on the leading strand with priming events on the lagging strand.     

Evidence for a Xer/dif system for chromosome resolution in archaea

Homologous recombination events between circular chromosomes, occurring during or after replication, can generate dimers that need to be converted to monomers prior to their segregation at cell division. In Escherichia coli, chromosome dimers are converted to monomers by two paralogous site-specific tyrosine recombinases of the Xer family (XerC/D). The Xer recombinases act at a specific dif site located in the replication termination region, assisted by the cell division protein FtsK. This chromosome resolution system has been predicted in most Bacteria and further characterized for some species. Archaea have circular chromosomes and an active homologous recombination system and should therefore resolve chromosome dimers. Most archaea harbour a single homologue of bacterial XerC/D proteins (XerA), but not of FtsK. Therefore, the role of XerA in chromosome resolution was unclear. Here, we have identified dif-like sites in archaeal genomes by using a combination of modeling and comparative genomics approaches. These sites are systematically located in replication termination regions. We validated our in silico prediction by showing that the XerA protein of Pyrococcus abyssi specifically recombines plasmids containing the predicted dif site in vitro. In contrast to the bacterial system, XerA can recombine dif sites in the absence of protein partners. Whereas Archaea and Bacteria use a completely different set of proteins for chromosome replication, our data strongly suggest that XerA is most likely used for chromosome resolution in Archaea.     

DNA replication in the archaea.

The archaeal DNA replication machinery bears striking similarity to that of eukaryotes and is clearly distinct from the bacterial apparatus. In recent years, considerable advances have been made in understanding the biochemistry of the archaeal replication proteins. Furthermore, a number of structures have now been obtained for individual components and higher-order assemblies of archaeal replication factors, yielding important insights into the mechanisms of DNA replication in both archaea and eukaryotes.