RESUMO
The three-dimensional organization of chromosomes can have a profound impact on their replication and expression. The chromosomes of higher eukaryotes possess discrete compartments that are characterized by differing transcriptional activities. Contrastingly, most bacterial chromosomes have simpler organization with local domains, the boundaries of which are influenced by gene expression. Numerous studies have revealed that the higher-order architectures of bacterial and eukaryotic chromosomes are dependent on the actions of structural maintenance of chromosomes (SMC) superfamily protein complexes, in particular, the near-universal condensin complex. Intriguingly, however, many archaea, including members of the genus Sulfolobus do not encode canonical condensin. We describe chromosome conformation capture experiments on Sulfolobus species. These reveal the presence of distinct domains along Sulfolobus chromosomes that undergo discrete and specific higher-order interactions, thus defining two compartment types. We observe causal linkages between compartment identity, gene expression, and binding of a hitherto uncharacterized SMC superfamily protein that we term "coalescin."
Assuntos
Proteínas de Ciclo Celular/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Cromossomos de Archaea/metabolismo , Sulfolobus/citologia , Sulfolobus/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas Cromossômicas não Histona/genética , Cromossomos de Archaea/genética , Replicação do DNA/genética , DNA Arqueal/metabolismo , Proteínas de Ligação a DNA/metabolismo , Expressão Gênica , Loci Gênicos/genética , Modelos Genéticos , Complexos Multiproteicos/metabolismo , Plasmídeos/genética , Ligação Proteica/genética , Transcrição GênicaRESUMO
BACKGROUND: In the last decade and a half it has been firmly established that a large number of proteins do not adopt a well-defined (ordered) structure under physiological conditions. Such intrinsically disordered proteins (IDPs) and intrinsically disordered (protein) regions (IDRs) are involved in essential cell processes through two basic mechanisms: the entropic chain mechanism which is responsible for rapid fluctuations among many alternative conformations, and molecular recognition via short recognition elements that bind to other molecules. IDPs possess a high adaptive potential and there is special interest in investigating their involvement in organism evolution. RESULTS: We analyzed 2554 Bacterial and 139 Archaeal proteomes, with a total of 8,455,194 proteins for disorder content and its implications for adaptation of organisms, using three disorder predictors and three measures. Along with other findings, we revealed that for all three predictors and all three measures (1) Bacteria exhibit significantly more disorder than Archaea; (2) plasmid-encoded proteins contain considerably more IDRs than proteins encoded on chromosomes (or whole genomes) in both prokaryote superkingdoms; (3) plasmid proteins are significantly more disordered than chromosomal proteins only in the group of proteins with no COG category assigned; (4) antitoxin proteins in comparison to other proteins, are the most disordered (almost double) in both Bacterial and Archaeal proteomes; (5) plasmidal proteins are more disordered than chromosomal proteins in Bacterial antitoxins and toxin-unclassified proteins, but have almost the same disorder content in toxin proteins. CONCLUSION: Our results suggest that while disorder content depends on genome and proteome characteristics, it is more influenced by functional engagements than by gene location (on chromosome or plasmid).
Assuntos
Archaea/genética , Proteínas Arqueais/química , Bactérias/genética , Proteínas de Bactérias/química , Proteínas Intrinsicamente Desordenadas/química , Plasmídeos/metabolismo , Cromossomos de Archaea/metabolismo , Cromossomos Bacterianos/metabolismo , Proteoma/metabolismo , Toxinas Biológicas/químicaRESUMO
DNA replication is fundamental to the propagation of all life on the planet. Remarkably, given the central importance for this process, two distinct core cellular DNA replication machineries have evolved. One is found in the bacterial domain of life and the other is present in Archaea and Eukarya. The archaeal machinery represents a simplified and presumably ancestral form of the eukaryotic DNA replication apparatus. As such, archaeal replication proteins have been studied extensively as models for their eukaryal counterparts. In addition, a number of archaea have been developed as model organisms. Accordingly, there has been a considerable increase in our knowledge of how archaeal chromosomes are replicated. It has become apparent that the majority of archaeal cells replicate their genomes from multiple origins per chromosome. Thus, at both organizational and mechanistic levels, archaeal DNA replication resembles that of eukarya. In this chapter, we will describe recent advances in our understanding of the basis of archaeal origin definition and how the archaeal initiator proteins recruit the replicative helicase to origins.
Assuntos
Archaea/enzimologia , Archaea/genética , DNA Helicases/metabolismo , Replicação do DNA , DNA Arqueal/biossíntese , Origem de Replicação , Proteínas Arqueais/metabolismo , Cromossomos de Archaea/genética , Cromossomos de Archaea/metabolismoRESUMO
The euryarchaeon Thermococcus kodakarensis is a well-characterized anaerobic hyperthermophilic heterotroph and due to the availability of genetic engineering systems it has become one of the model organisms for studying Archaea. Despite this prominent role among the Euryarchaeota, no data about the ploidy level of this species is available. While polyploidy has been shown to exist in various Euryarchaeota, especially Halobacteria, the chromosome copy number of species belonging to one of the major orders within that phylum, i.e., the Thermococcales (including Thermococcus spp. and Pyrococcus spp.), has never been determined. This prompted us to investigate the chromosome copy number of T. kodakarensis. In this study, we demonstrate that T. kodakarensis is polyploid with a chromosome copy number that varies between 7 and 19 copies, depending on the growth phase. An apparent correlation between the presence of histones and polyploidy in Archaea is observed.
Assuntos
Cromossomos de Archaea/genética , Thermococcus/genética , Cromossomos de Archaea/metabolismo , Thermococcus/metabolismoRESUMO
Architectural DNA proteins play important roles in the chromosomal DNA organization and global gene regulation in living cells. However, physiological functions of some DNA-binding proteins from archaea remain unclear. Recently, several abundant DNA-architectural proteins including histones, Alba, and TrmBL2 have been identified in model euryarchaeon Thermococcus kodakarensis. Although histones and Alba proteins have been previously characterized, the DNA binding properties of TrmBL2 and its interplay with the other major architectural proteins in the chromosomal DNA organization and gene transcription regulation remain largely unexplored. Here, we report single-DNA studies showing that at low ionic strength (<300 mM KCl), TrmBL2 binds to DNA largely in non-sequence-specific manner with positive cooperativity, resulting in formation of stiff nucleoprotein filamentous patches, whereas at high ionic strength (>300 mM KCl) TrmBL2 switches to more sequence-specific interaction, suggesting the presence of high affinity TrmBL2-filament nucleation sites. Furthermore, in vitro assays indicate the existence of DNA binding competition between TrmBL2 and archaeal histones B from T. kodakarensis, which can be strongly modulated by DNA supercoiling and ionic strength of surrounding solution. Overall, these results advance our understanding of TrmBL2 DNA binding properties and provide important insights into potential functions of architectural proteins in nucleoid organization and gene regulation in T. kodakarensis.
Assuntos
Proteínas Arqueais/metabolismo , Cromossomos de Archaea/metabolismo , DNA Arqueal/metabolismo , DNA Super-Helicoidal/metabolismo , Histonas/metabolismo , Proteínas Repressoras/metabolismo , Thermococcus/metabolismo , Proteínas Arqueais/genética , Cromossomos de Archaea/genética , DNA Arqueal/genética , DNA Super-Helicoidal/genética , Histonas/genética , Proteínas Repressoras/genética , Thermococcus/genéticaRESUMO
Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (cas) genes constitute the adaptive immune system in bacteria and archaea. Although the CRISPR-Cas systems have been hypothesized to encode potential toxins, no experimental data supporting the hypothesis are available in the literature. In this work, we provide the first experimental evidence for the presence of a toxin gene in the type I-A CRISPR system of hyperthermophilic archaeon Sulfolobus. csa5, under the control of its native promoter in a shuttle vector, could not be transformed into CRISPR-deficient mutant Sulfolobus solfataricus Sens1, demonstrating a strong toxicity in the cells. A single-amino-acid mutation destroying the intersubunit bridge of Csa5 attenuated the toxicity, indicative of the importance of Csa5 oligomerization for its toxicity. In line with the absence of Csa5 toxicity in S. solfataricus InF1 containing functional CRISPR systems, the expression of csa5 is repressed in InF1 cells. Induced from the arabinose promoter in Sens1 cells, Csa5 oligomers resistant to 1% SDS co-occur with chromosome degradation and cell death, reinforcing the connection between Csa5 oligomerization and its toxicity. Importantly, a rudivirus was shown to induce Csa5 expression and the formation of SDS-resistant Csa5 oligomers in Sulfolobus cells. This demonstrates that the derepression of csa5 and the subsequent Csa5 oligomerization take place in native virus-host systems. Thus, csa5 is likely to act as a suicide gene under certain circumstances to inhibit virus spreading.
Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Sulfolobus solfataricus/metabolismo , Apoptose , Western Blotting , Proteínas Associadas a CRISPR/genética , Cromossomos de Archaea/metabolismo , DNA Arqueal/metabolismo , Técnicas de Inativação de Genes , Mutação/genética , Regiões Promotoras Genéticas/genética , Rudiviridae/patogenicidade , Sulfolobus solfataricus/crescimento & desenvolvimento , Sulfolobus solfataricus/virologiaRESUMO
Replication of the main chromosome in the halophilic archaeon Haloferax volcanii was recently reported to continue despite deletion of all active replication origins. Equally surprising, the deletion strain grew faster than the parent strain. It was proposed that origin-less H. volcanii duplicate their chromosomes via recombination-dependent replication. Here, we recall our present knowledge of this mode of chromosome replication in different organisms. We consider the likelihood that it accounts for the viability of H. volcanii deleted for its main specific replication origins, as well as possible alternative interpretations of the results. The selective advantages of having defined chromosome replication origins are discussed from a functional and evolutionary perspective.
Assuntos
Cromossomos de Archaea/metabolismo , Haloferax volcanii/metabolismo , Origem de Replicação , Sequência de Bases , Replicação do DNA , Recombinação Genética/genéticaAssuntos
Archaea/genética , Bactérias/genética , Segregação de Cromossomos , Cromossomos de Archaea/metabolismo , Cromossomos Bacterianos/metabolismo , Archaea/fisiologia , Cromossomos de Archaea/ultraestrutura , Cromossomos Bacterianos/ultraestrutura , Modelos Biológicos , Células Procarióticas/fisiologiaRESUMO
Knowledge of the chromosome biology of archaeal species has grown considerably in the last 15 years, since the publication of the first full archaeal genome sequences. A number of model organisms have been studied, revealing a striking variety of mechanisms and modes of genome duplication and segregation. While clear sequence relationships between archaeal and eukaryotic replication proteins are well known, some archaea also seem to possess organizational parameters for replication and segregation that reveal further striking parallels to eukaryotes.
Assuntos
Archaea/fisiologia , Cromossomos de Archaea/metabolismo , Replicação do DNA , Archaea/genética , Proteínas Arqueais/metabolismo , Segregação de CromossomosRESUMO
Until recently little was known about the cell cycle parameters and division mechanisms of archaeal organisms. Although this is still the case for the majority of archaea, significant advances have been made in some model species. The information that has been gleaned thus far points to a remarkable degree of diversity within the archaeal domain of life. More specifically, members of distinct phyla have very different chromosome copy numbers, replication control systems and even employ distinct machineries for cell division.
Assuntos
Archaea/fisiologia , Ciclo Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Archaea/metabolismo , Modelos BiológicosRESUMO
The powerful combination of genetic and biochemical analysis has provided many key insights into the structure and function of the chromosomal DNA replication machineries of bacterial and eukaryotic cells. In contrast, in the archaea, biochemical studies have dominated, mainly due to the absence of efficient genetic systems for these organisms. This situation is changing, however, and, in this regard, the genetically tractable haloarchaea Haloferax volcanii and Halobacterium sp. NRC-1 are emerging as key models. In the present review, I give an overview of the components of the replication machinery in the haloarchaea, with particular emphasis on the protein factors presumed to travel with the replication fork.
Assuntos
Cromossomos de Archaea/metabolismo , Replicação do DNA , Proteínas Arqueais/metabolismo , DNA/metabolismo , Proteínas Nucleares/metabolismo , Origem de ReplicaçãoRESUMO
The minichromosome maintenance (MCM) complex is thought to function as the replicative helicase in archaea and eukarya. The structure of the single MCM protein homologue from the archaeon Methanothermobacter thermautotrophicus is not yet clear, and hexameric, heptameric, octameric, and dodecameric structures, open rings, and filamentous structures have been reported. Using a combination of biochemical and structural analysis, it is shown here that the M. thermautotrophicus MCM helicase is active as a hexamer.
Assuntos
Proteínas Arqueais/química , DNA Helicases/química , Methanobacteriaceae/enzimologia , Proteínas Arqueais/metabolismo , Cromossomos de Archaea/metabolismo , DNA Helicases/metabolismo , Estrutura Quaternária de Proteína/fisiologia , Relação Estrutura-AtividadeRESUMO
In order to reveal functional properties of recombination involving short ssDNAs in hyperthermophilic archaea, we evaluated oligonucleotide-mediated transformation (OMT) in Sulfolobus acidocaldarius and Escherichia coli as a function of the molecular properties of the ssDNA substrates. Unmodified ssDNAs as short as 20-22 nt yielded recombinants in both organisms, as did longer DNAs forming as few as 2-5 base pairs on one side of the genomic mutation. The two OMT systems showed similar responses to certain end modifications of the oligonucleotides, but E. coli was found to require a 5' phosphate on 5'-limited ssDNA whereas this requirement was not evident in S. acidocaldarius. The ability of both E. coli and S. acidocaldarius to incorporate short, mismatched ssDNAs into their genomes raises questions about the biological significance of this capability, including its phylogenetic distribution among microorganisms and its impact on genome stability. These questions seem particularly relevant for S. acidocaldarius, as this archaeon has natural competence for OMT, encodes no MutSL homologues and thrives under environmental conditions that accelerate DNA decomposition.
Assuntos
Escherichia coli/genética , Oligonucleotídeos/química , Recombinação Genética , Sulfolobus acidocaldarius/genética , Proteínas Virais/metabolismo , Bacteriófago lambda/genética , Sequência de Bases , Cromossomos de Archaea/genética , Cromossomos de Archaea/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA Arqueal/genética , DNA Arqueal/metabolismo , DNA Bacteriano/genética , DNA Bacteriano/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Escherichia coli/metabolismo , Escherichia coli/virologia , Dados de Sequência Molecular , Oligonucleotídeos/síntese química , Oligonucleotídeos/genética , Oligonucleotídeos/metabolismo , Especificidade por Substrato , Sulfolobus acidocaldarius/metabolismo , Transformação Genética , Proteínas Virais/genéticaRESUMO
DNA base composition asymmetry is at the basis of numerous in silico methods for the detection of the origin and terminus of replication in prokaryotes. However, most of these methods are unable to identify the evolutionary mechanisms that cause the base composition asymmetry. In prokaryotic chromosomes, due to the tendency for coorientation between replication and transcription, compositional biases that discriminate the leading strand from the lagging strand can be produced by 2 superposing mechanisms: replication-associated mutation bias and coding sequence-associated bias (such as transcription-related mutational processes or selective pressures on codon usage). We propose here a new method for the analysis of nucleotide composition asymmetry that allows the decoupling of replication-related and coding sequence-related mechanisms. This method is inspired by a recent work (Nikolaou and Almirantis 2005) that proposed an artificial chromosomal rearrangement meant to create a perfect gene orientation bias. We show that the study of nucleotide skews on the artificially rearranged chromosomes is a powerful means to assess the contributions of the 2 types of mechanisms in generating the base composition asymmetry. We applied our method to all completely sequenced prokaryotic chromosomes available. Our results confirm that in most species the replication mechanism has an important effect on base composition asymmetry but also that it has different impacts on GC and AT skews. We also analyzed the variability in AT skew direction encountered in prokaryotes. In disagreement with a recent report (Worning et al. 2006), we find that the polymerase-alpha subunits encoded in a genome are not sufficient to predict the sign of the AT skew on its leading strand for replication.
Assuntos
Composição de Bases , Replicação do DNA , DNA , Sequência de Bases , Cromossomos de Archaea/genética , Cromossomos de Archaea/metabolismo , Cromossomos Bacterianos/genética , Cromossomos Bacterianos/metabolismo , DNA/genética , DNA/metabolismo , Modelos Genéticos , Células ProcarióticasRESUMO
The primary candidate for the eukaryotic replicative helicase is the MCM2-7 complex, a hetero-oligomer formed by six AAA+ paralogous polypeptides. A simplified model for structure-function studies is the homo-oligomeric orthologue from the archaeon Methanothermobacter thermoautotrophicus. The crystal structure of the DNA-interacting N-terminal domain of this homo-oligomer revealed a double hexamer in a head-to-head configuration; single-particle electron microscopy studies have shown that the full-length protein complex can form both single and double rings, in which each ring can consist of a cyclical arrangement of six or seven subunits. Using single-particle techniques and especially multivariate statistical symmetry analysis, we have assessed the changes in stoichiometry that the complex undergoes when treated with various nucleotide analogues or when binding a double-stranded DNA fragment. We found that the binding of nucleotides or of double-stranded DNA leads to the preferred formation of double-ring structures. Specifically, the protein complex is present as a double heptamer when treated with a nucleotide analogue, but it is rather found as a double hexamer when complexed with double-stranded DNA. The possible physiological role of the various stoichiometries of the complex is discussed in the light of the proposed mechanisms of helicase activity.
Assuntos
Proteínas Arqueais/metabolismo , Cromossomos de Archaea/metabolismo , DNA Helicases/química , DNA Helicases/metabolismo , Difosfato de Adenosina/farmacologia , Proteínas Arqueais/química , Proteínas Arqueais/isolamento & purificação , Proteínas Arqueais/ultraestrutura , Cromossomos de Archaea/química , DNA/metabolismo , DNA Helicases/genética , Escherichia coli/genética , Methanobacteriaceae/enzimologia , Modelos Biológicos , Nucleotídeos/metabolismo , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Especificidade por Substrato , Transformação GenéticaRESUMO
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.
Assuntos
Cromossomos de Archaea/genética , Cromossomos de Archaea/metabolismo , Replicação do DNA , DNA Arqueal/biossíntese , DNA Arqueal/genética , Sulfolobus solfataricus/genética , Sulfolobus solfataricus/metabolismo , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , DNA Primase/química , DNA Primase/genética , DNA Primase/metabolismo , DNA Arqueal/química , Substâncias Macromoleculares , Modelos Moleculares , Conformação de Ácido Nucleico , Conformação Proteica , Origem de ReplicaçãoRESUMO
Methanobacterium thermoautotrophicum minichromosome maintenance complex (mtMCM), a cellular replicative helicase, is a useful model for the more complex eukaryotic MCMs. Biochemical and crystallographic evidence indicates that mtMCM assembles as a double hexamer (dHex), but previous electron microscopy studies reported only the presence of single heptamers or single hexamers (Pape, T., Meka, H., Chen, S., Vicentini, G., Van Heel, M., and Onesti, S. (2003) EMBO Rep. 4, 1079-1083; Yu, X., VanLoock, M. S., Poplawski, A., Kelman, Z., Xiang, T., Tye, B. K., and Egelman, E. H. (2002) EMBO Rep. 3, 792-797). Here we present the first three-dimensional electron microscopy reconstruction of the full-length mtMCM dHex in which two hexamers contact each other via the structurally well defined N-terminal domains. The dHex has obvious side openings that resemble the side channels of LTag (large T antigen). 6-fold and 7-fold rings were observed in the same mtMCM preparation, but we determined that assembly as a double ring favors 6-fold structures. Additionally, open rings were also detected, which suggests a direct mtMCM loading mechanism onto DNA.
Assuntos
Cromossomos de Archaea/metabolismo , DNA Helicases/química , DNA Helicases/genética , Methanobacterium/enzimologia , Polimorfismo Genético , Cromossomos de Archaea/química , DNA Bacteriano/metabolismo , Escherichia coli/genética , Microscopia Eletrônica , Modelos Moleculares , Dobramento de Proteína , Proteínas Recombinantes/químicaRESUMO
Sac7d, a small, abundant, sequence-general DNA-binding protein from the hyperthermophilic archaeon Sulfolobus acidocaldarius, causes a single-step sharp kink in DNA (approximately 60 degrees) via the intercalation of both Val26 and Met29. These two amino acids were systematically changed in size to probe their effects on DNA kinking. Eight crystal structures of five Sac7d mutant-DNA complexes have been analyzed. The DNA-binding pattern of the V26A and M29A single mutants is similar to that of the wild-type, whereas the V26A/M29A protein binds DNA without side chain intercalation, resulting in a smaller overall bending (approximately 50 degrees). The M29F mutant inserts the Phe29 side chain orthogonally to the C2pG3 step without stacking with base pairs, inducing a sharp kink (approximately 80 degrees). In the V26F/M29F-GCGATCGC complex, Phe26 intercalates deeply into DNA bases by stacking with the G3 base, whereas Phe29 is stacked on the G15 deoxyribose, in a way similar to those used by the TATA box-binding proteins. All mutants have reduced DNA-stabilizing ability, as indicated by their lower T m values. The DNA kink patterns caused by different combinations of hydrophobic side chains may be relevant in understanding the manner by which other minor groove-binding proteins interact with DNA.
Assuntos
Proteínas Arqueais/química , Proteínas de Ligação a DNA/química , DNA/química , Proteínas Arqueais/genética , Proteínas Arqueais/metabolismo , Sítios de Ligação , Cromossomos de Archaea/metabolismo , DNA/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Temperatura Alta , Modelos Moleculares , Mutação , Conformação de Ácido NucleicoRESUMO
In the present paper, we report that a Cdc6 (cell-division control)-like factor from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (referred to as SsoCdc6-2) has a modular organization of its biological functions. A reliable model of the SsoCdc6-2 three-dimensional structure was built up, based on the significant sequence identity with the Pyrobaculum aerophylum Cdc6 (PaeCdc6), whose crystallographic structure is known. This allowed us to design two truncated forms of SsoCdc6-2: the DeltaC (residues 1-297, molecular mass 35 kDa) and the DeltaN (residues 298-400, molecular mass 11 kDa) proteins. The DeltaC protein contains the nucleotide-binding Rossmann fold and the Sensor-2 motif (Domains I and II in the PaeCdc6 structure), and retains the ability to bind and hydrolyse ATP. On the other hand, the DeltaN protein contains the C-terminal WH (winged helix)-fold (Domain III), and is able to bind DNA molecules and to inhibit the DNA helicase activity of the SsoMCM (mini-chromosome maintenance) complex, although with lesser efficiency with respect to the full-sized SsoCdc6-2. These results provide direct biochemical evidence that the Cdc6 WH-domain is responsible for DNA-binding and inhibition of MCM DNA helicase activity.
Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Saccharomyces cerevisiae/genética , Sulfolobus/química , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/farmacologia , Processamento Alternativo/genética , Sequência de Aminoácidos , Proteínas Arqueais/biossíntese , Proteínas Arqueais/genética , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/metabolismo , Cromossomos de Archaea/metabolismo , DNA/metabolismo , DNA Helicases/metabolismo , DNA de Cadeia Simples/metabolismo , Proteínas de Ligação a DNA/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosforilação , Ligação Proteica/efeitos dos fármacos , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Homologia de Sequência de Aminoácidos , Sulfolobus/enzimologiaRESUMO
The genome of Halobacterium salinarum encodes four proteins of the structural maintenance of chromosomes (SMC) protein superfamily. Two proteins form a novel subfamily and are named 'SMC-like proteins of H. salinarum' (Sph1 and Sph2). Northern blot analyses revealed that sph1 and hp24, the adjacent gene, are solely transcribed in exponentially growing, but not in stationary phase, cells. A synchronization procedure was developed, which makes use of the DNA polymerase inhibitor aphidicolin and leads to highly synchronous cultures. It allowed us for the first time to study cell cycle-dependent transcription in an archaeon. The sph1 transcript was found to be highly cell cycle regulated, with its maximal accumulation around the time of septum formation. The Sph1 protein level was also elevated at that time, but a basal protein level was found throughout the cell cycle. The hp24 transcript was sharply upregulated about 1 h before sph1 and had already declined at the time of sph1 induction. These and additional transcript patterns revealed that precisely controlled transcriptional regulation is involved in haloarchaeal cell cycle progression. A DNA staining protocol was developed, which opened the possibility of following the dynamic intracellular localization of haloarchaeal nucleoids using synchronized cultures. After an initial dispersed localization, the nucleoid is condensed at mid-cell. Subsequently, DNA is rapidly transported to the 1/4 and 3/4 positions. All staining patterns were also observed in untreated exponentially growing cells, excluding synchronization artifacts. The Sph1 concentration is elevated when segregation of the new chromosomes is nearly complete; therefore, it is proposed to play a role in a late step of replication, e.g. DNA repair, similar to eukaryotic Rad18 proteins.