Regulating DNA Replication in Eukarya

DNA replication is tightly controlled in eukaryotic cells to ensure that an exact copy of the genetic material is inherited by both daughter cells. Oscillating waves of cyclin-dependent kinase (CDK) and anaphase-promoting complex/cyclosome (APC/C) activities provide a binary switch that permits the replication of each chromosome exactly once per cell cycle. Work from several organisms has revealed a conserved strategy whereby inactive replication complexes are assembled onto DNA during periods of low CDK and high APC activity but are competent to execute genome duplication only when these activities are reversed. Periods of high CDK and low APC/C serve an essential function by blocking reassembly of replication complexes, thereby preventing rereplication. Higher eukaryotes have evolved additional CDK-independent mechanisms for preventing rereplication.The Eukarya include a wide spectrum of organisms, with genome sizes ranging from ∼10⁷ bp in yeasts to ∼10¹² bp in protozoa. Rapid duplication of large genomes is achieved by distribution of the genetic material across several chromosomes. Each of these chromosomes initiates replication from sites called replication origins, which must fire no more than once per cell cycle to ensure a single error-free copy of the genome. Generating replication forks from an origin more than once leads to rereplication, an event that creates multiple copies of a single genomic region within a single cell. This leads to gene amplification and promotes genome instability (Green et al. 2010), a phenomenon observed in many human cancers (Lengauer et al. 1998). The process of genome duplication is therefore under stringent control to ensure that few, if any, defects are transmitted from one generation to the next.     

Computational analysis reveals abundance of potential glycoproteins in Archaea, Bacteria and Eukarya

Glycosylation is the most common type of post-translational modification (PTM) and is known to affect protein stability, folding and activity. Inactivity of enzymes mediating glycosylation can result in serious disorders including colon cancer and brain disorders. Out of five main types of glycosylation, N-linked glycosylation is most abundant and characterized by the addition of a sugar group to an Asparagine residue at the N-X-S/T motif. Enzyme mediating such transfer is known as oligosaccharyl transferase (OST). It has been hypothesized before that a significant number of proteins serve as glycoproteins. In this study, we used programming implementations of Python to statistically quantify the representation of glycoproteins by scanning all the available proteome sequence data at ExPASy server for the presence of glycoproteins and also the enzyme which plays critical role in glycosylation i.e. OST. Our results suggest that more than 50% of the proteins carry N-X-S/T motif i.e. they could be potential glycoproteins. Furthermore, approximately 28-36% (1/3) of proteins possesses signature motifs which are characteristic features of enzyme OST. Quantifying this bias individually reveals that both the number of proteins tagged with N-X-S/T motif and the average number of motifs per protein is significantly higher in case of eukaryotes when compared to prokaryotes. In the light of these results we conclude that there is a significant bias in the representation of glycoproteins in the proteomes of all species and is manifested substantially in eukaryotes and claim for glycosylation to be the most common and ubiquitous PTM in cells, especially in eukaryotes.     

From volcanic origins of chemoautotrophic life to Bacteria, Archaea and Eukarya

The theory of a chemoautotrophic origin of life in a volcanic iron-sulphur world postulates a pioneer organism at sites of reducing volcanic exhalations. The pioneer organism is characterized by a composite structure with an inorganic substructure and an organic superstructure. Within the surfaces of the inorganic substructure iron, cobalt, nickel and other transition metal centres with sulphido, carbonyl and other ligands were catalytically active and promoted the growth of the organic superstructure through carbon fixation, driven by the reducing potential of the volcanic exhalations. This pioneer metabolism was reproductive by an autocatalytic feedback mechanism. Some organic products served as ligands for activating catalytic metal centres whence they arose. The unitary structure-function relationship of the pioneer organism later gave rise to two major strands of evolution: cellularization and emergence of the genetic machinery. This early phase of evolution ended with segregation of the domains Bacteria, Archaea and Eukarya from a rapidly evolving population of pre-cells. Thus, life started with an initial, direct, deterministic chemical mechanism of evolution giving rise to a later, indirect, stochastic, genetic mechanism of evolution and the upward evolution of life by increase of complexity is grounded ultimately in the synthetic redox chemistry of the pioneer organism.     

Serpins in Unicellular Eukarya, Archaea, and Bacteria: Sequence Analysis and Evolution

Most serpins irreversibly inactivate specific serine proteinases of the chymotrypsin family. Inhibitory serpins are unusual proteins in that their native structure is metastable, and rapid conversion to a relaxed state is required to trap target enzymes in a covalent complex. The evolutionary origin of the serpin fold is unresolved, and while serpins in animals are known to be involved in the regulation of a remarkable diversity of metabolic processes, the physiological functions of homologues from other phyla are unknown. Addressing these questions, here we analyze serpin genes identified in unicellular eukaryotes: the green alga Chlamydomonas reinhardtii, the dinoflagellate Alexandrium tamarense, and the human pathogens Entamoeba spp., Eimera tenella, Toxoplasma gondii, and Giardia lamblia. We compare these sequences to others, particularly those in the complete genome sequences of Archaea, where serpins were found in only 4 of 13 genera, and Bacteria, in only 9 of 56 genera. The serpins from unicellular organisms appear to be phylogenetically distinct from all of the clades of higher eukaryotic serpins. Most of the sequences from unicellular organisms have the characteristics of inhibitory serpins, and where multiple serpin genes are found in one genome, variability is displayed in the region of the reactive-center loop important for specificity. All the unicellular eukaryotic serpins have large hydrophobic or positively charged residues at the putative P₁ position. In contrast, none of the prokaryotic serpins has a residue of these types at the predicted P₁ position, but many have smaller, neutral residues. Serpin evolution is discussed.[Reviewing Editor: Dr. Peer Bork]     

Common evolutionary origins of mechanosensitive ion channels in Archaea, Bacteria and cell-walled Eukarya

The ubiquity of mechanosensitive (MS) channels triggered a search for their functional homologs in Archaea. Archaeal MS channels were found to share a common ancestral origin with bacterial MS channels of large and small conductance, and sequence homology with several proteins that most likely function as MS ion channels in prokaryotic and eukaryotic cell-walled organisms. Although bacterial and archaeal MS channels differ in conductive and mechanosensitive properties, they share similar gating mechanisms triggered by mechanical force transmitted via the lipid bilayer. In this review, we suggest that MS channels of Archaea can bridge the evolutionary gap between bacterial and eukaryotic MS channels, and that MS channels of Bacteria, Archaea and cell-walled Eukarya may serve similar physiological functions and may have evolved to protect the fragile cellular membranes in these organisms from excessive dilation and rupture upon osmotic challenge.     

Not just for Eukarya anymore: protein glycosylation in Bacteria and Archaea

Of the many post-translational modifications proteins can undergo, glycosylation is the most prevalent and the most diverse. Today, it is clear that both N-glycosylation and O-glycosylation, once believed to be restricted to eukaryotes, also transpire in Bacteria and Archaea. Indeed, prokaryotic glycoproteins rely on a wider variety of monosaccharide constituents than do those of eukaryotes. In recent years, substantial progress in describing the enzymes involved in bacterial and archaeal glycosylation pathways has been made. It is becoming clear that enhanced knowledge of bacterial glycosylation enzymes may be of therapeutic value, while the demonstrated ability to introduce bacterial glycosylation genes into Escherichia coli represents a major step forward in glyco-engineering. A better understanding of archaeal protein glycosylation provides insight into this post-translational modification across evolution as well as protein processing under extreme conditions. Here, we discuss new structural and biosynthetic findings related to prokaryotic protein glycosylation, until recently a neglected topic.     

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.     

Common domains in the initiators of DNA replication in Bacteria,Archaea and Eukarya: combined structural,functional and phylogenetic perspectives

Although DNA replication is the universal process for the transmission of genetic information in all living organisms, until very recently evidence was lacking for a related structure and function in the proteins (initiators) that trigger replication in the three 'Life Domains' (Bacteria, Archaea and Eukarya). In this article new data concerning the presence of common features in the initiators of chromosomal replication in bacteria, archaea and eukaryotes are reviewed. Initiators are discussed in the light of: (i) The structure and function of their conserved ATPases Associated with various cellular Activities (AAA+) and winged-helix domains. (ii) The nature of the macromolecular assemblies that they constitute at the replication origins. (iii) Their possible phylogenetic relationship, attempting to sketch the essentials of a hypothetical DNA replication initiator in the micro-organism proposed to be the ancestor of all living cells.     

Geobiological analysis using whole genome-based tree building applied to the Bacteria, Archaea, and Eukarya

We constructed genomic trees based on the presence and absence of families of protein‐encoding genes observed in 55 prokaryotic and five eukaryotic genomes. There are features of the genomic trees that are not congruent with typical rRNA phylogenetic trees. In the bacteria, for example, Deinococcus radiodurans associates with the Gram‐positive bacteria, a result that is also seen in some other phylogenetic studies using whole genome data. In the Archaea, the methanogens plus Archaeoglobus form a united clade and the Euryarchaeota are divided with the two Thermoplasma genomes and Halobacterium sp. falling below the Crenarchaeota. While the former appears to be an accurate representation of methanogen‐relatedness, the misplacement of Halobacterium may be an artefact of parsimony. These results imply the last common ancestor of the Archaea was not a methanogen, leaving sulphur reduction as the most geochemically plausible metabolism for the base of the archaeal crown group. It also suggests that methanogens were not a component of the Earth's earliest biosphere and that their origin occurred sometime during the Archean. In the Eukarya, the parsimony analysis of five Eukaryotes using the Crenarchaeota as an outgroup seems to counter the Ecdysozoa hypothesis, placing Caenorhabditis elegans (Nematoda) below the common ancestor of Drosophila melanogaster (Arthropoda) and Homo sapiens (Chordata) even when efforts are made to counter the possible effects of a faster rate of sequence evolution for the C. elegans genome. Further analysis, however, suggests that the gene loss of ‘animal’ genes is highest in C. elegans and is obscuring the relationships of these organisms.     

Evolution of DNA Replication Protein Complexes in Eukaryotes and Archaea

Background

The replication of DNA in Archaea and eukaryotes requires several ancillary complexes, including proliferating cell nuclear antigen (PCNA), replication factor C (RFC), and the minichromosome maintenance (MCM) complex. Bacterial DNA replication utilizes comparable proteins, but these are distantly related phylogenetically to their archaeal and eukaryotic counterparts at best.

Methodology/Principal Findings

While the structures of each of the complexes do not differ significantly between the archaeal and eukaryotic versions thereof, the evolutionary dynamic in the two cases does. The number of subunits in each complex is constant across all taxa. However, they vary subtly with regard to composition. In some taxa the subunits are all identical in sequence, while in others some are homologous rather than identical. In the case of eukaryotes, there is no phylogenetic variation in the makeup of each complex—all appear to derive from a common eukaryotic ancestor. This is not the case in Archaea, where the relationship between the subunits within each complex varies taxon-to-taxon. We have performed a detailed phylogenetic analysis of these relationships in order to better understand the gene duplications and divergences that gave rise to the homologous subunits in Archaea.

Conclusion/Significance

This domain level difference in evolution suggests that different forces have driven the evolution of DNA replication proteins in each of these two domains. In addition, the phylogenies of all three gene families support the distinctiveness of the proposed archaeal phylum Thaumarchaeota.     

Giant viruses coexisted with the cellular ancestors and represent a distinct supergroup along with superkingdoms Archaea, Bacteria and Eukarya

ABSTRACT: BACKGROUND: The discovery of giant viruses with genome and physical size comparable to cellular organisms, remnants of protein translation machinery and virus-specific parasites (virophages) have raised intriguing questions about their origin. Evidence advocates for their inclusion into global phylogenomic studies and their consideration as a distinct and ancient form of life. RESULTS: Here we reconstruct phylogenies describing the evolution of proteomes and protein domain structures of cellular organisms and double-stranded DNA viruses with medium-to-very-large proteomes (giant viruses). Trees of proteomes define viruses as a 'fourth supergroup' along with superkingdoms Archaea, Bacteria, and Eukarya. Trees of domains indicate they have evolved via massive and primordial reductive evolutionary processes. The distribution of domain structures suggests giant viruses harbor a significant number of protein domains including those with no cellular representation. The genomic and structural diversity embedded in the viral proteomes is comparable to the cellular proteomes of organisms with parasitic lifestyles. Since viral domains are widespread among cellular species, we propose that viruses mediate gene transfer between cells and crucially enhance biodiversity. CONCLUSIONS: Results call for a change in the way viruses are perceived. They likely represent a distinct form of life that either predated or coexisted with the last universal common ancestor (LUCA) and constitute a very crucial part of our planet's biosphere.     

Genome DNA Sequence Variation, Evolution, and Function in Bacteria and Archaea

Comparative genomics has revealed that variations in bacterial and archaeal genome DNA sequences cannot be explained by only neutral mutations. Virus resistance and plasmid distribution systems have resulted in changes in bacterial and archaeal genome sequences during evolution. The restriction-modification system, a virus resistance system, leads to avoidance of palindromic DNA sequences in genomes. Clustered, regularly interspaced, short palindromic repeats (CRISPRs) found in genomes represent yet another virus resistance system. Comparative genomics has shown that bacteria and archaea have failed to gain any DNA with GC content higher than the GC content of their chromosomes. Thus, horizontally transferred DNA regions have lower GC content than the host chromosomal DNA does. Some nucleoid-associated proteins bind DNA regions with low GC content and inhibit the expression of genes contained in those regions. This form of gene repression is another type of virus resistance system. On the other hand, bacteria and archaea have used plasmids to gain additional genes. Virus resistance systems influence plasmid distribution. Interestingly, the restriction-modification system and nucleoid-associated protein genes have been distributed via plasmids. Thus, GC content and genomic signatures do not reflect bacterial and archaeal evolutionary relationships.     

Histones and nucleosomes in Archaea and Eukarya: a comparative analysis

Archaeal histones from mesophilic, thermophilic, and hyperthermophilic members of the Euryarchaeota have primary sequences, the histone fold, tertiary structures, and dimer formation in common with the eukaryal nucleosome core histones H2A, H2B, H3, and H4. Archaeal histones form nucleoprotein complexes in vitro and in vivo, designated archaeal nucleosomes, that contain histone tetramers and protect approximately 60 base pairs of DNA from nuclease digestion. Based on the sequence and structural homologies and experimental data reviewed here, archaeal nucleosomes appear similar, and may be homologous in evolutionary terms and function, to the structure at the center of the eukaryal nucleosome formed by the histone (H3+H4)₂ tetramer. Received: January 22, 1998 / Accepted: February 16, 1998     

Serine/Threonine Protein Kinases from Bacteria,Archaea and Eukarya Share a Common Evolutionary Origin Deeply Rooted in the Tree of Life

The main family of serine/threonine/tyrosine protein kinases present in eukarya was defined and described by Hanks et al. in 1988 (Science, 241, 42–52). It was initially believed that these kinases do not exist in bacteria, but extensive genome sequencing revealed their existence in many bacteria. For historical reasons, the term “eukaryotic-type kinases” propagated in the literature to describe bacterial members of this protein family. Here, we argue that this term should be abandoned as a misnomer, and we provide several lines of evidence to support this claim. Our comprehensive phylostratigraphic analysis suggests that Hanks-type kinases present in eukarya, bacteria and archaea all share a common evolutionary origin in the lineage leading to the last universal common ancestor (LUCA). We found no evidence to suggest substantial horizontal transfer of genes encoding Hanks-type kinases from eukarya to bacteria. Moreover, our systematic structural comparison suggests that bacterial Hanks-type kinases resemble their eukaryal counterparts very closely, while their structures appear to be dissimilar from other kinase families of bacterial origin. This indicates that a convergent evolution scenario, by which bacterial kinases could have evolved a kinase domain similar to that of eukaryal Hanks-type kinases, is not very likely. Overall, our results strongly support a monophyletic origin of all Hanks-type kinases, and we therefore propose that this term should be adopted as a universal name for this protein family.     

Interdomain Conjugal Transfer of DNA from Bacteria to Archaea

Escherichia coli transforms the methanogenic archaeon Methanococcus maripaludis at frequencies ranging from 0.2 × 10⁻⁶ to 2 × 10⁻⁶ per recipient cell. Transformation requires cell-to-cell contact, oriT, and tra functions, is insensitive to DNase I, and otherwise displays hallmarks of conjugation.Conjugal transfer of DNA involves a specific set of transfer (tra) functions that mediate the mobilization of DNA containing an origin of transfer (oriT) from a donor to a recipient in a process requiring cell-to-cell contact (9). While conjugation is often very efficient between members of a given species or genus, it can also occur at a lower efficiency between phylogenetically distant microorganisms with structurally distinct cell surfaces. Escherichia coli, for example, mediates conjugal transfer of DNA to such diverse bacterial recipients as cyanobacteria (23), spirochetes (14), and a variety of Gram-positive bacteria (17, 22); E. coli even mediates conjugal DNA transfer to members of the domain Eukarya, such as to Saccharomyces cerevisiae (6) and mammalian (20) cells. Because of its broad range of potential recipients, conjugation has proven to be a valuable genetic tool (11) and may be an important mechanism of horizontal gene transfer and a driver of genome evolution (7). Conjugation-like DNA transfer has also been demonstrated in members of the domain Archaea (5, 15). However, conjugation between Bacteria and Archaea has not been demonstrated, despite the observation that many whole-genome sequences of Archaea harbor DNA that appears to be of bacterial origin (7).To investigate whether conjugation can occur between Bacteria and Archaea, the RP4 (IncPα group) conjugal-transfer system was used to attempt to mobilize DNA from E. coli to the anaerobic, methanogenic archaeon Methanococcus maripaludis strain S2 (21). The RP4 system was selected because previous work demonstrated that this plasmid supports the transfer of DNA from E. coli to phylogenetically distant recipients, including yeast (3) and mammalian (20) cells. Additionally, E. coli has been shown to successfully conjugate with strictly anaerobic bacterial strains (22). M. maripaludis was chosen as a recipient because it has growth parameters similar to those of E. coli and has readily available selectable markers (1). For all the experiments described, M. maripaludis was grown in liquid or solid (excluding cysteine) McCas medium (12), supplemented with 2.5 μg/ml puromycin (Pur) where appropriate, using standard anaerobic techniques (2). All plating for conjugation experiments, except for determination of viable-E. coli cell counts, was performed in an anaerobic chamber (Coy, Grass Lake, MI) with an atmosphere of 5:5:90 H₂-CO₂-N₂. E. coli was grown in Difco LB medium (Becton-Dickinson, Sparks, MD) supplemented where appropriate with 50 μg/ml kanamycin sulfate (Kan) and ampicillin (Amp).To interrogate conjugal DNA transfer between E. coli and M. maripaludis, a set of vectors that either contained or lacked cis-acting sites required for mobilization by RP4 transfer functions were constructed (Table ​(Table1).1). Each of these vectors contained a Pur resistance (Pur^r) gene cassette (pac) (4) flanked by ∼0.5 kb DNA homologous to regions 5′ and 3′ of the M. maripaludis nrpR gene (nrpR::pac), which allows for selection by Pur in M. maripaludis and provides sites for homologous recombination into the nrpR locus of the M. maripaludis chromosome. This construct was selected because it has previously been used to transform M. maripaludis to Pur resistance by recombination into the nrpR locus using a polyethylene glycol (PEG)-mediated transformation protocol (10, 18). After it was demonstrated that plasmids of the appropriate genotypes support conjugation from donor strain E. coli S17-1, which contains the RP4 trans-acting transfer (tra) functions on the chromosome via an integrated RP4-2-Tc::Mu-Km::Tn7 cassette (16), to E. coli recipient cells (Table ​(Table1;1; see also the supplemental material), we investigated whether these same donor strains could support DNA transfer to M. maripaludis.

TABLE 1.

Transformation of M. maripaludis by E. coli

Analysis of genomic tRNA sets from Bacteria, Archaea, and Eukarya points to anticodon-codon hydrogen bonds as a major determinant of tRNA compositional variations

Analysis of 100 complete sets of the cytoplasmic elongator tRNA genes from Bacteria, Archaea, and Eukarya pointed to correspondences between types of anticodon and composition of the rest of the tRNA body. The number of the hydrogen bonds formed between the complementary nucleotides in the anticodon-codon duplex appeared as a major quantitative parameter determining covariations in all three domains of life. Our analysis has supported and advanced the "extended anticodon" concept that is based on the argument that the decoding performance of the anticodon is enhanced by selection of a matching anticodon stem-loop sequence, as reported by Yarus in 1982. In addition to the anticodon stem-loop, we have found covariations between the anticodon nucleotides and the composition of the distant regions of their respective tRNAs that include dihydrouridine (D) and thymidyl (T) stem-loops. The majority of the covariable tRNA positions were found at the regions with the increased dynamic potential--such as stem-loop and stem-stem junctions. The consistent occurrences of the covariations on the multigenomic level suggest that the number and pattern of the hydrogen bonds in the anticodon-codon duplex constitute a major factor in the course of translation that is reflected in the fine-tuning of the tRNA composition and structure.     

Failsafe Mechanisms Couple Division and DNA Replication in Bacteria

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The universal ancestor and the ancestors of Archaea and Bacteria were anaerobes whereas the ancestor of the Eukarya domain was an aerobe

The use of an oxyphobic index (OI) based on the propensity of amino acids to enter more frequently the proteins of anaerobes makes it possible to make inferences on the environment in which the last universal common ancestor (LUCA) lived. The reconstruction of the ancestral sequences of proteins using a method based on maximum likelihood and their attribution by means of the OI to the set of aerobe or anaerobe sequences has led to the following conclusions: the LUCA was an anaerobic 'organism', as were the ancestors of Archaea and Bacteria, whereas the ancestor of Eukarya was an aerobe. These observations seem to falsify the hypothesis that the LUCA was an aerobe and help to identify better the environment in which the first organisms lived.