DNA replication, similar in all systems, involves initiation, elongation, and termination. The replication of DNA, beginning at the origins of replication present on the circular chromosomes, requires initiator proteins. The recruitment of additional proteins by way of the initiator proteins allows the separation of the circular DNA and results in the formation of a bubble.
The relationship between the three domains is of central importance for understanding the origin of life. Within prokaryotes, archaeal cell structure is most similar to that of Gram-positive bacteria, largely because both have a single lipid bilayer and usually contain a thick sacculus of varying chemical composition.
Archaea and Gram-positive bacteria also share conserved indels in a number of important proteins, such as Hsp70 and glutamine synthetase I. Gupta has proposed that the Archaea evolved from Gram-positive bacteria in response to antibiotic selection pressure. This is suggested by the observation that archaea are resistant to a wide variety of antibiotics that are primarily produced by Gram-positive bacteria, and that these antibiotics primarily act on the genes that distinguish Archaea from Bacteria.
The evolution of Archaea in response to antibiotic selection, or any other competitive selective pressure, could also explain their adaptation to extreme environments such as high temperature or acidity as the result of a search for unoccupied niches to escape from antibiotic-producing organisms; Cavalier-Smith has made a similar suggestion.
Archaea possess genes and several metabolic pathways that are more closely related to those of eukaryotes than prokaryotes. The evolutionary relationship between archaea and eukaryotes remains unclear. Aside from the similarities in cell structure and function that are discussed below, many genetic trees group the two.
Archaea and other domains : Phylogenetic tree showing the relationship between the Archaea and other domains of life. Eukaryotes are colored red, archaea green and bacteria blue. Complicating factors include claims that the relationship between eukaryotes and the archaeal phylum Crenarchaeota is closer than the relationship between the Euryarchaeota and the phylum Crenarchaeota, and the presence of archaean-like genes in certain bacteria, such as Thermotoga maritima , from horizontal gene transfer.
The leading hypothesis is that the ancestor of the eukaryotes diverged early from the Archaea, and that eukaryotes arose through fusion of an archaean and eubacterium, which became the nucleus and cytoplasm.
This explains various genetic similarities but runs into difficulties when it comes to explaining cell structure. Despite this visual similarity to bacteria, archaea possess genes and several metabolic pathways that are more closely-related to those of eukaryotes, notably the enzymes involved in transcription and translation. Archaea exhibit a great variety of chemical reactions in their metabolism and use many sources of energy.
Despite the bacterial-like structure of archaeal replication origins, archaea use eukaryotic-type replication machinery Robinson and Bell, , indicating that archaea may adopt eukaryotic-like mechanisms to control replication proteins and thus replication initiation.
Interestingly, genome-wide transcription mapping indicated that serine—threonine protein kinases show cyclic induction in Sulfolobus species, indicating that regulatory factors similar to eukaryotic cyclin-dependent kinase CDK complexes may be present in archaea Lundgren and Bernander, In addition, as almost all replication origins are dependent on Cdc6 proteins, conformational changes of Cdc6 proteins may play important roles in coordinating replication initiation at different origins within a cell.
Although considerable diversity of replication origins has been observed in haloarchaea, comparison analysis revealed a conserved replication origin, oriC1 , which is positioned in the main chromosome of all analyzed haloarchaeal genomes Coker et al.
In addition, gene order analysis found that genes around oriC1 are highly syntenic among haloarchaea Figure 2 ; Capes et al. Notably, other studies Robinson et al.
Variations were observed in oriC1 homologs from different archaeal phyla, which may contribute to the adaptability of archaea to different extreme environments. The conserved oriC1 origin of replication in sequenced haloarchaeal genomes.
The oriC1 context region was mapped as shown in the sequenced haloarchaea. The colored boxed arrows represent different genes as follows: GTP-binding protein gbp , teal , initiator protein cdc6 , red , signal sequence peptidase sec , yellow and DNA-directed DNA polymerase polA , blue. The inverted ORB elements are indicated by small triangles. Multiple replication origins along with their adjacent cdc6 genes appear to be mosaics of distinct replicator—initiator systems. A comparison between Aeropyrum and Sulfolobus origins suggested that the capture of extrachromosomal elements accounts for replicon evolution Robinson and Bell, In particular, it has been proposed that the three replication origins of the Sulfolobus species arose by the integration of extrachromosomal elements into a single-origin ancestral chromosome oriC1-cdc , and the acquisition of oriC3-whiP occurred prior to the integration of oriC2-cdc Samson et al.
In addition, comparative analyses of the selected replication origins suggested that different evolutionary mechanisms, including ancestral conservation and coupled acquisition and deletion events, may account for the current mosaics of multiple replication origins in the haloarchaeal genomes.
Importantly, a comparative genomic analysis of two Haloarcula species, Haloarcula hispanica and Haloarcula marismortui , revealed that the species-specific origins are located in extremely variable regions, suggesting that these novel origins were recently acquired, via either integration into the chromosome or rearrangement of extrachromosomal elements Wu et al. Further work may focus on comparisons of replication origins from closely related species to reveal the dynamics of origin evolution and whether origin evolution alters the mode of genomic replication.
To date, the number of archaea with mapped replication origins is still limited, which to some extent has affected us to get a panoramic view of the generality and evolution of replication origins in archaea.
In addition to the mapping of replication origins, the development of prediction algorithms for replication origins in archaeal genomes and the construction of databases with these predicted origins Gao et al. Fortunately, the rapid increase in the number of complete archaeal genomic sequences that are publically available will promote our studies of archaeal replication origins. In addition, the control and coordination of replication initiation at multiple origins in archaea is far less understood.
The multireplicon structure of haloarchaeal genomes allows for precise control and coordination of replication initiation at multiple origins. As the chromosome and extrachromosomal elements within a haloarchaeon are generally different sizes and have different copy numbers Breuert et al.
In addition, the coordination of multiple origins may play important roles in maintaining the multireplicon structure of haloarchaeal genomes. As most replication origins are dependent on Cdc6 proteins in haloarchaea excluding the origins of small plasmids , we propose that the coordination of replication initiation at different origins may be obtained by conformational changes of Cdc6 proteins via an ATP-ADP binary switch, which has recently been proposed for chromosome replication in S.
Thus, more exhaustive work should be taken into account to uncover the control and coordination of the replication initiation from multiple origins, either on the same chromosome or from different genetic elements, in haloarchaeal multireplicon genomes.
The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. This work was partially supported by grants from the National Natural Science Foundation of China , , Aparicio, O. Genes Dev. Berquist, B. An archaeal chromosomal autonomously replicating sequence element from an extreme halophile, Halobacterium sp.
Breuert, S. Regulated polyploidy in halophilic archaea. Capaldi, S. Nucleic Acids Res. Capes, M. The information transfer system of halophilic archaea. Plasmid 65, 77— Coker, J.
Multiple replication origins of Halobacterium sp. Dueber, E. Molecular determinants of origin discrimination by Orc1 initiators in archaea. Replication origin recognition and deformation by a heterodimeric archaeal Orc1 complex. Science , — Duggin, I. Chromosome replication dynamics in the archaeon Sulfolobus acidocaldarius.
Egan, E. Distinct replication requirements for the two Vibrio cholerae chromosomes. Cell , — Gao, F. DoriC 5. DoriC: a database of oriC regions in bacterial genomes. Bioinformatics 23, — Ori-Finder: a web-based system for finding oriC s in unannotated bacterial genomes.
BMC Bioinformatics Gaudier, M. Grainge, I. Biochemical analysis of a DNA replication origin in the archaeon Aeropyrum pernix. Hawkins, M. Accelerated growth in the absence of DNA replication origins. Nature , — Hyrien, O. Jacob, F. Spring Harb. CrossRef Full Text. Kelman, L. Multiple origins of replication in archaea. Trends Microbiol. Kitagawa, R. Negative control of replication initiation by a novel chromosomal locus exhibiting exceptional affinity for Escherichia coli DnaA protein.
Leonard, A. DNA replication origins. And it also suggested that an active NER pathway exists in archaea. Clearly, additional experiments are now needed to better understand the role of Tko Hef in NER and, more generally, to further dissect the pathway responsible for archaeal Nucleotide Excision Repair. In contrast, the deletion of hef in H. Methods used for exposure of H. A possible explanation for these phenotypic differences is that NER proteins in Thermococcus and Haloferax species are very different.
In fact H. To further investigate the role of Hef in H. Among several combinations tested, we demonstrated that Hef was essential for viability in the absence of the Holliday junctions HJs resolvase Hjc.
Holliday junctions are four-way branched DNA structures formed during homologous recombination strand exchange and recombination-dependent replication restart. HJs resolvases are found in bacteria, archaea and eukarya, although they are not evolutionary related. Hjc is conserved throughout archaea. The single deletion of hjc gene in H. Kodakarensis cells did not affect growth rate, DNA repair or recombination [ 32 , 65 ].
In this scenario Hef could use its helicase activity on arrested replication forks to process them into four-way DNA structures that can be resolved by its nuclease activity. This scenario was compatible with the in vitro studies described above. Indeed, point mutations inactivating the helicase activity Hvo Hef-K48A or the nuclease activity Hvo Hef-DA of Hvo Hef resulted in the same phenotype observed in the absence of the entire protein.
This nicely demonstrated that both helicase and nuclease activities of Hef were required for fulfilling its role in the absence of the Hjc resolvase [ 32 ]. To test the hypothesis that Hef and Hjc were both acting as HJs resolvase, we deleted hef or hjc in a strain carrying a radA deletion.
In absence of RadA recombinase, HJs are no longer formed by homologous recombination so that deleting HJs resolvases should not have any affect. Indeed cells deleted for both radA and hjc were phenotypically similar to cells only deleted for radA. This observation was consistent with a role of Hjc in the resolution of HJs formed by RadA-mediated strand exchange during homologous recombination. However, radA gene could not be deleted in hef -deleted cells [ 32 ], strongly reflecting that functional roles of Hjc and Hef were distinct.
This observation also suggested that Hef was required for cell viability in absence of recombination. Replication restart is one possible pathway. But how could we obtain more detailed information on functional roles of Hef if hef -deleted mutant strains hardly shows any phenotype or cannot be combined with other deletions? We decided to develop tools to allow dynamic localization of fluorescently-labelled Hef proteins in living Haloferax volcanii cells.
Growth phenotypes of H. It is encoded by a single polypeptide containing the chromophore. After translation of the protein, an autocatalytic process involving oxygen has to take place within the chromophore. Once active, the GFP has a major excitation peak at a wavelength of nm and an emission peak at nm. A deep understanding of the protein has enabled the development of several GFP variants with modified spectral properties [ 71 , 72 ]. Such approaches can reveal key features of proteins in vivo to complete our understanding of pathways, as illustrated for NER pathway in mammalian cells [ 74 ], but their use in archaeal cells has been rather limited until recently.
GFP has been used to investigate proteasome-dependant proteolysis and protein levels in H. Because GFPs variant can differ not only by their fluorescence properties but also by their maturation rate of the fluorophore, temperature and pH stability or oligomeric state for instance, one has to carefully choose the variants that optimally fit the lifestyle of the organism being studied. We have recently used GFP-fusions to investigate protein localization and behaviour in archaeal cells for the first time.
These studies were performed using the halophile H. This variant has mutations increasing solubility Phe99Ser, MetThr and Val63Ala as well as a mutation in one of the three amino acids of the chromophore Ser65Thr that redshifts the absorption maximum to nm without changing the emission properties of the protein [ 78 , 79 ].
The resulting fusion protein was expressed under physiological expression levels and conditions from the native chromosomal locus of the hef gene [ 33 ]. Whether GFP-fused Hef proteins remained functional was then tested by comparing cells deleted for hef with cells expressing the hef :: gfp allele. Because we were interested in the localization of Hef in response to replication arrests, we exposed cells to aphidicolin APD , an antibiotic that inhibits DNA synthesis in halophilic archaea [ 80 ], thus arresting replication forks.
Exposing hef -deleted cells to increasing concentrations of APD decreased cell viability, showing that indeed Hef is involved in the genomic stability upon replication arrest. Such decrease in cell viability was not observed with cells expressing GFP-fused Hef. We then observed the localisation of Hef::GFP proteins by fluorescence microscopy, comparing cells exposed to APD to non-treated control samples.
Towards this goal, a drop of cells was spotted on an agarose slice placed on a glass slide. After allowing this drop to dry, the agarose pad was covered with a cover-slip for cell imaging studies using a wield-field microscope to visualize a large number of individual cells. Then fluorescence imaging was performed exciting at nm and collecting at nm. In order not to lose any information, fluorescence images were acquired at different focal planes on the z-axis.
Consecutive slices of cells in focus were then selected and used to perform a maximum intensity z-projection. At each pixel, the highest fluorescence signal was kept when comparing the selected images. This maximum intensity z-projection resulted into a two-dimensional picture where the maximal fluorescence signals from different focal planes were recorded Figure 4.
Schematic representation of fluorescence signal analysed in cells. A Representation of a cell with fluorescence foci and the different focal planes used for imaging.
B Representation of fluorescence signal in each focal plane. C Resulting image after projection of the maximum fluorescence signal at each pixel for the four focal planes. Different imaging parameters were optimized to detect cells and fluorescence foci within cells using automatic thresholds to avoid user-bias. This approch allowed thousands of cells to be analysed in each condition tested, providing extremely high statistical power.
Using such approach, we have shown that Hef::GFP proteins formed fluorescence foci even under normal growth condition, in the absence of any DNA damaging agents. The number of these foci was significantly increased from two to four foci per cell in response to aphidicolin exposure. We also observed that the number of foci per individual cell changed significantly.
While the majority of cells had one or two foci in normal growth conditions, a higher proportion of cells having more than two foci was observed upon APD exposure Figure 5. In vivo localization of GFP-labeled Hef in response to aphidicolin exposure. A total of spots within control cells and spots within APD-treated cells were analyzed. B Average cell surface of hef :: gfp cells in response to increasing concentrations of aphidicolin.
C Mean number of GFP-Hef labeled fluorescence foci per cell in response to increasing concentrations of aphidicolin. D Relative frequency of number of foci per individual cell. All error bars represent SD. From [ 33 ]. APD exposure. We have shown using other DNA damaging agents that increased cell size and number of foci were specific to APD treatment, suggesting that indeed Hvo Hef is recruited at arrested replication forks brought about by addition of aphidicolin.
These experiments were performed using a confocal microscope on cells immobilized on a poly-D-lysine coated cover-slip. In FRAP experiments a region of interest was photobleached in a cell.
The speed of fluorescence recovery in that region was then measured, reflecting the diffusion of Hef::GFP fluorescent molecules arriving from the non-photobleached region of the cell. In control cells no aphidicolin , one major population of Hef::GFP diffusing molecules was observed. From the fit of the recovery curve we obtained the recovery constant, allowing then the apparent two-dimensional diffusion rate of Hef::GFP to be estimated at 0.
This appeared markedly lower than expected for Hef dimer, as revealed by analytical ultracentrifugation experiments on purified Hvo Hef further indicating that Hef has a peculiar elongated shape in solution. Several possibilities may explain this limited diffusion. But FRAP experiments performed on cells exposed to aphidicolin revealed an additional, even more slowly-diffusing population that was clearly induced by APD treatment Figure 6.
Fluorescence Recovery After Photobleaching experiments to study the dynamic localization of GFP-labeled Hef molecules at fluorescence foci. FRAP regions are shown by white circles.
Time after photobleaching in seconds. B Fluorescence recovery curve averaged for 9 control cells. C Fluorescence recovery curve averaged for 8 aphidicolin treated-cells. These analyses were performed on one hundred images taken every 2 seconds, and cell regions including and excluding fluorescence foci were compared. Fluctuation of fluorescence intensity per pixel was then used to determine the number of diffusing molecules and their brightness. This information can then be used to deduce changes in the oligomeric state of the fluorescent molecules.
Interestingly, hjc deletion had effect neither on cell size nor on the number of foci per cell in normal growth condition as well as in response to APD treatment. These observations showed that recruitment of Hef to arrested replication forks was not increased in the absence of the alternative pathway involving Hjc and RadA , suggesting that Hef is recruited at arrested replication forks even in the presence of the alternative HR-dependent pathway.
We also noted that in eukarya recent studies have indicated that FANCM proteins can prevent homologous recombination [ 82 - 85 ].
0コメント