Les ADN topoisomérases du crenarchaeon hyperthermophile Sulfolobus solfataricus (régulateurs du métabolisme de l'ADN ?)

Les ADN topoisomérases sont des enzymes capables de moduler la torsion de la double hélice d ADN afin de rendre compatible sa topologie avec les différents processus cellulaires impliquant l ADN. Les hyperthermophiles possèdent au moins une topoisomérase particulière, la reverse gyrase qui est constituée à la fois d un domaine topoisomérase IA etd un domaine hélicase de type SF2. Mon sujet de thèse a eu pour objectif de déterminer principalement l implication des ADN topoisomérases IA dans les différents processus cellulaires de Sulfolobus solfataricus. Ce crenarchaeon hyperthermophile possède, en plus, d une ADN topoisomérase de type II (Topo VI), trois ADN-topoisomérases IA dont une classique (TopA) et deux reverse gyrases (TopR1 et TopR2). Notre approche a permis d estimer, pour la première fois, le nombre de TopR1 et de TopR2 par cellule en fonction des différentes conditions testées. L étude des variations quantitatives des ADN topoisomérases a clairement mis en évidence que TopR1 et TopR2 sont régulées différemment ce qui renforce l hypothèse d une spécialisation de leurs fonctions. Nous avons ainsi montré que TopR1 est responsable du maintien de l homéostasie du surenroulement de l ADN. Si la Topo VI de par son activité antagoniste est impliquée dansce même contrôle homéostatique, elle ne fait pas l objet d une régulation quantitative. De plus, nous avons mis en évidence que TopR1 était liée à la vie à haute température. Enfin, nos résultats suggèrent que TopR2 serait pour sa part impliquée dans la stabilité des génomes. L identification des partenaires protéiques respectifs des quatre ADN topoisomérases de S. solfataricus permettra d avoir une vision globale des réseaux de régulation permettant derésoudre les différentes des contraintes topologiques générées au cours de la vie de cet hyperthermophile.DNA topoisomerases act in all DNA metabolism processes to control the DNA topology. Hyperthermophiles possess at least a particular topoisomerase, the reverse gyrase composed of a DNA topoisomerase IA domain and a helicase SF2 domain within the same polypeptide. The general objective of my thesis was to determine the involvement of each DNA topoisomerase in different cellular processes of S. solfataricus. This hyperthermophilic crenarchaeon possesses in addition to a type II DNA topoisomerase (Topo VI), three DNA topoisomerases IA : a classical one (TopA) and two reverse gyrases (TopR1 and TopR2). Our experimental approach allowed to estimate for the first time the number of TopR1 and TopR2 per cell in relation to different conditions. The study of quantitative variations of each DNA topoisomerase clearly showed that TopR1 and TopR2 are differently regulated suggesting that they are involved in distinct cellular processes. Indeed, we showed that TopR1 is the main actor of the homeostatic control of the DNA supercoiling. If the Topo VI with its antogonistic activity is involved in this homeostatic control, there is no regulation at the level of protein quantity. In addition we evidenced that TopR1 is somehow linked to the life at high temperature. Our results suggest that TopR2 is involved in genome stability. The identification of the respective potential partners of the four DNA topoisomerases of S. solfataricus will allow to get a more detailed understanding of the DNA topology regulation during the hyperthermophilic life style.EVRY-Bib. électronique (912289901) / SudocSudocFranceF

DNA topoisomerases of the crenarchaeon hyperthermophile Sulfolobus solfataricus : regulators of DNA metabolism ?

Les ADN topoisomérases sont des enzymes capables de moduler la torsion de la double hélice d’ADN afin de rendre compatible sa topologie avec les différents processus cellulaires impliquant l’ADN. Les hyperthermophiles possèdent au moins une topoisomérase particulière, la reverse gyrase qui est constituée à la fois d’un domaine topoisomérase IA etd’un domaine hélicase de type SF2. Mon sujet de thèse a eu pour objectif de déterminer principalement l’implication des ADN topoisomérases IA dans les différents processus cellulaires de Sulfolobus solfataricus. Ce crenarchaeon hyperthermophile possède, en plus, d’une ADN topoisomérase de type II (Topo VI), trois ADN-topoisomérases IA dont une « classique » (TopA) et deux reverse gyrases (TopR1 et TopR2). Notre approche a permis d’estimer, pour la première fois, le nombre de TopR1 et de TopR2 par cellule en fonction des différentes conditions testées. L’étude des variations quantitatives des ADN topoisomérases a clairement mis en évidence que TopR1 et TopR2 sont régulées différemment ce qui renforce l’hypothèse d’une spécialisation de leurs fonctions. Nous avons ainsi montré que TopR1 est responsable du maintien de l’homéostasie du surenroulement de l’ADN. Si la Topo VI de par son activité antagoniste est impliquée dansce même contrôle homéostatique, elle ne fait pas l’objet d’une régulation quantitative. De plus, nous avons mis en évidence que TopR1 était liée à la vie à haute température. Enfin, nos résultats suggèrent que TopR2 serait pour sa part impliquée dans la stabilité des génomes. L’identification des partenaires protéiques respectifs des quatre ADN topoisomérases de S. solfataricus permettra d’avoir une vision globale des réseaux de régulation permettant derésoudre les différentes des contraintes topologiques générées au cours de la vie de cet hyperthermophile.DNA topoisomerases act in all DNA metabolism processes to control the DNA topology. Hyperthermophiles possess at least a particular topoisomerase, the reverse gyrase composed of a DNA topoisomerase IA domain and a helicase SF2 domain within the same polypeptide. The general objective of my thesis was to determine the involvement of each DNA topoisomerase in different cellular processes of S. solfataricus. This hyperthermophilic crenarchaeon possesses in addition to a type II DNA topoisomerase (Topo VI), three DNA topoisomerases IA : a classical one (TopA) and two reverse gyrases (TopR1 and TopR2). Our experimental approach allowed to estimate for the first time the number of TopR1 and TopR2 per cell in relation to different conditions. The study of quantitative variations of each DNA topoisomerase clearly showed that TopR1 and TopR2 are differently regulated suggesting that they are involved in distinct cellular processes. Indeed, we showed that TopR1 is the main actor of the homeostatic control of the DNA supercoiling. If the Topo VI with its antogonistic activity is involved in this homeostatic control, there is no regulation at the level of protein quantity. In addition we evidenced that TopR1 is somehow linked to the life at high temperature. Our results suggest that TopR2 is involved in genome stability. The identification of the respective potential partners of the four DNA topoisomerases of S. solfataricus will allow to get a more detailed understanding of the DNA topology regulation during the hyperthermophilic life style

The DNA Methylome of the Hyperthermoacidophilic Crenarchaeon Sulfolobus acidocaldarius

DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius

Essential role of two tyrosines and two tryptophans on the photoprotection activity of the Orange Carotenoid Protein

Photosynthetic organisms have developed photoprotective mechanisms to protect themselves from lethal high light intensities. One of these mechanisms involves the dissipation of excess absorbed light energy into heat. In cyanobacteria, light activation of a soluble carotenoid protein, the Orange Carotenoid Protein (OCP), binding a keto carotenoid. is the key inducer of this mechanism. Blue-green light absorption triggers structural changes within the carotenoid and the protein, leading to the conversion of a dark orange form into a red active form. Here we report the role in photoconversion and photoprotection of individual conserved tyrosines and tryptophans surrounding the rings of the carotenoid. Our results demonstrate that the interaction between the keto group of the carotenoid and Tyr201 and Trp288 is essential for OCP photoactivity. In addition, these amino acids are responsible for carotenoid affinity and specificity. We have already demonstrated that the aromatic character of Tyr44 and Trp110 interacting with the hydroxyl ring is critical. Here we show that the replacement of Tyr44 by Ser affects the stability of the red form avoiding its accumulation at any temperature, while Trp110Ser is affected in the energy necessary to the orange to red conversion and in the interaction with the antenna. Collectively our data support the idea that the red form is essential for photoprotection but not sufficient. Specific conformational changes occurring in the protein seem to be critical to the events leading to energy dissipation. (C) 2010 Elsevier B.V. All rights reserved

DNA-Binding Properties of a Novel Crenarchaeal Chromatin-Organizing Protein in Sulfolobus acidocaldarius

In archaeal microorganisms, the compaction and organization of the chromosome into a dynamic but condensed structure is mediated by diverse chromatin-organizing proteins in a lineage-specific manner. While many archaea employ eukaryotic-type histones for nucleoid organization, this is not the case for the crenarchaeal model species Sulfolobus acidocaldarius and related species in Sulfolobales, in which the organization appears to be mostly reliant on the action of small basic DNA-binding proteins. There is still a lack of a full understanding of the involved proteins and their functioning. Here, a combination of in vitro and in vivo methodologies is used to study the DNA-binding properties of Sul12a, an uncharacterized small basic protein conserved in several Sulfolobales species displaying a winged helix–turn–helix structural motif and annotated as a transcription factor. Genome-wide chromatin immunoprecipitation and target-specific electrophoretic mobility shift assays demonstrate that Sul12a of S. acidocaldarius interacts with DNA in a non-sequence specific manner, while atomic force microscopy imaging of Sul12a–DNA complexes indicate that the protein induces structural effects on the DNA template. Based on these results, and a contrario to its initial annotation, it can be concluded that Sul12a is a novel chromatin-organizing protein

Transcriptional and translational dynamics underlying heat shock response in the thermophilic crenarchaeon Sulfolobus acidocaldarius

International audienceHigh-temperature stress is critical for all organisms and induces a profound cellular response. For Crenarchaeota, little information is available on how heat shock affects cellular processes and on how this response is regulated. We set out to study heat shock response in the thermoacidophilic model crenarchaeon Sulfolobus acidocaldarius, which thrives in volcanic hot springs and has an optimal growth temperature of 75°C. Pulse-labeling experiments demonstrated that a temperature shift to 86°C induces a drastic reduction of the transcriptional and translational activity, but that RNA and protein neosynthesis still occurs. By combining RNA sequencing and mass spectrometry, an integrated mapping of the transcriptome and proteome was performed. This revealed that heat shock causes an immediate change in the gene expression profile, with RNA levels of half of the genes being affected, followed by a more subtle reprogramming of the protein landscape. Functional enrichment analysis indicated that nearly all cellular processes are affected by heat shock. A limited correlation was observed in the differential expression on the RNA and protein level, suggesting a prevalence of post-transcriptional and post-translational regulation. Furthermore, promoter sequence analysis of heat shock regulon genes demonstrated the conservation of strong transcription initiation elements for highly induced genes, but an absence of a conserved protein-binding motif. It is, therefore, hypothesized that histone-lacking archaea such as Sulfolobales use an evolutionarily ancient regulatory mechanism that relies on temperature-responsive changes in DNA organization and compaction induced by the action of nucleoid-associated proteins, as well as on enhanced recruitment of initiation factors