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Maria Ciaramella

Publications and source records attributed to Maria Ciaramella.

10 recordsLinked to original sources
PubMed2006

Lack of strand-specific repair of UV-induced DNA lesions in three genes of the archaeon Sulfolobus solfataricus.

In all organisms, specialized systems are devoted to repair of DNA lesions induced by exposure to UV light. In both Eucarya and Bacteria, UV-induced pyrimidine dimers in the transcribed strand of active genes are repaired at a faster rate compared to the non-transcribed strand and the rest of the genome. Preferential repair of transcribed strands requires the Transcription-Repair Coupling Factor in Escherichia coli and the CSA and CSB proteins in humans. These factors are needed for coupling of transcription to nucleotide excision repair (NER), a major pathway for repair of UV-induced lesions. Whereas transcription-coupled NER (TC-NER) is an evolutionary conserved process, not all active genes show preferential repair of transcribed strands. The existence of a NER pathway in the Archaea has not been demonstrated directly, yet it is suggested by the presence and properties of homologues of NER nucleases and helicases. However, none of the proteins responsible for the lesion recognition steps or for TC-NER has been found in archaeal genomes. Moreover, the kinetics of gene or strand-specific repair has never been investigated in any organism of this domain. We have analysed the kinetics of repair of UV-induced DNA damage in the transcribed and non-transcribed strands of three genes of the hyperthermophilic archaeon Sulfolobus solfataricus. We found that in all three genes the two strands are repaired with the same efficiency with each other and with the genome in general, thus providing no evidence of strand bias or transcription coupling of the repair process in the genes analysed. Further studies will be required to test the existence of a transcription-coupled repair pathway in other archaeal genes and to elucidate the mechanism of UV lesion recognition and repair in Archaea.

Bacteria↗
PubMed2006

Selective degradation of reverse gyrase and DNA fragmentation induced by alkylating agent in the archaeon Sulfolobus solfataricus.

Reverse gyrase is a peculiar DNA topoisomerase, specific of hyperthermophilic Archaea and Bacteria, which has the unique ability of introducing positive supercoiling into DNA molecules. Although the function of the enzyme has not been established directly, it has been suggested to be involved in DNA protection and repair. We show here that the enzyme is degraded after treatment of Sulfolobus solfataricus cells with the alkylating agent MMS. MMS-induced reverse gyrase degradation is highly specific, since (i) neither hydroxyurea (HU) nor puromycin have a similar effect, and (ii) topoisomerase VI and two chromatin components are not degraded. Reverse gyrase degradation does not depend on protein synthesis. Experiments in vitro show that direct exposure of cell extracts to MMS does not induce reverse gyrase degradation; instead, extracts from MMS-treated cells contain some factor(s) able to degrade the enzyme in extracts from control cells. In vitro, degradation is blocked by incubation with divalent metal chelators, suggesting that reverse gyrase is selectively degraded by a metal-dependent protease in MMS-treated cells. In addition, we find a striking concurrence of extensive genomic DNA degradation and reverse gyrase loss in MMS-treated cells. These results support the hypothesis that reverse gyrase plays an essential role in DNA thermoprotection and repair in hyperthermophilic organisms.

Alkylating Agents↗
PubMed2005

Functional interaction of reverse gyrase with single-strand binding protein of the archaeon Sulfolobus.

Reverse gyrase is a unique hyperthermophile-specific DNA topoisomerase that induces positive supercoiling. It is a modular enzyme composed of a topoisomerase IA and a helicase domain, which cooperate in the ATP-dependent positive supercoiling reaction. Although its physiological function has not been determined, it can be hypothesized that, like the topoisomerase-helicase complexes found in every organism, reverse gyrase might participate in different DNA transactions mediated by multiprotein complexes. Here, we show that reverse gyrase activity is stimulated by the single-strand binding protein (SSB) from the archaeon Sulfolobus solfataricus. Using a combination of in vitro assays we analysed each step of the complex reverse gyrase reaction. SSB stimulates all the steps of the reaction: binding to DNA, DNA cleavage, strand passage and ligation. By co-immunoprecipitation of cell extracts we show that reverse gyrase and SSB assemble a complex in the presence of DNA, but do not make stable protein-protein interactions. In addition, SSB stimulates reverse gyrase positive supercoiling activity on DNA templates associated with the chromatin protein Sul7d. Furthermore, SSB enhances binding and cleavage of UV-irradiated substrates by reverse gyrase. The results shown here suggest that these functional interactions may have biological relevance and that the interplay of different DNA binding proteins might modulate reverse gyrase activity in DNA metabolic pathways.

Archaeal Proteins↗
PubMed2005

Another extreme genome: how to live at pH 0.

Many Archaea live under conditions that challenge the physico-chemical limits to life: low or high temperature, extremes of pH, elevated pressure and high salt concentration. A recent paper reports the genome sequence of another record-setting archaeon, Picrophilus torridus, that thrives at 65 degrees C and pH 0. The genomic sequence provides several hints of the mechanisms used for adaptation to such hostile environment, but most secrets remain hidden and await further analysis to be disclosed.

Cell Membrane↗
PubMed2004

Reverse gyrase recruitment to DNA after UV light irradiation in Sulfolobus solfataricus.

Induction of DNA damage triggers a complex biological response concerning not only repair systems but also virtually every cell function. DNA topoisomerases regulate the level of DNA supercoiling in all DNA transactions. Reverse gyrase is a peculiar DNA topoisomerase, specific to hyperthermophilic microorganisms, which contains a helicase and a topoisomerase IA domain that has the unique ability to introduce positive supercoiling into DNA molecules. We show here that reverse gyrase of the archaean Sulfolobus solfataricus is mobilized to DNA in vivo after UV irradiation. The enzyme, either purified or in cell extracts, forms stable covalent complexes with UV-damaged DNA in vitro. We also show that the reverse gyrase translocation to DNA in vivo and the stabilization of covalent complexes in vitro are specific effects of UV light irradiation and do not occur with the intercalating agent actinomycin D. Our results suggest that reverse gyrase might participate, directly or indirectly, in the cell response to UV light-induced DNA damage. This is the first direct evidence of the recruitment of a topoisomerase IA enzyme to DNA after the induction of DNA damage. The interaction between helicase and topoisomerase activities has been previously proposed to facilitate aspects of DNA replication or recombination in both Bacteria and Eukarya. Our results suggest a general role of the association of such activities in maintaining genome integrity and a mutual effect of DNA topology and repair.

DNA Damage↗
PubMed2003

Transcriptional response to DNA damage in the archaeon Sulfolobus solfataricus.

Exposure of cells to DNA-damaging agents triggers a complex biological response involving cell cycle arrest and modulation of gene expression. Genomic sequencing has revealed the presence of archaeal genes homologous to components of the eucaryal nucleotide excision repair (NER) pathway, which is involved in the repair of ultraviolet (UV) light-induced DNA damage. However, the events involved in the cell response to UV irradiation and their regulation have not been studied in Archaea. We show here that UV radiation induces the formation of cyclobutane pyrimidine dimers (CPDs) in the hyperthermophilic archaeon Sulfolobus solfataricus, and that these lesions are efficiently repaired in vivo in the dark, suggesting that a NER pathway is active. DNA damage is a signal for concomitant growth arrest and transcriptional induction of the NER genes XPF, XPG and XPB. The cell response to UV irradiation includes transcriptional regulation of genes encoding two DNA binding proteins involved in chromosome dynamics. Moreover, several of these genes are also strongly induced by the intercalating agent actinomycin D. Thus, response to DNA damage in S.solfataricus has features essentially conserved in all three domains of life.

Archaeal Proteins↗
PubMed2002

Ionic network at the C-terminus of the beta-glycosidase from the hyperthermophilic archaeon Sulfolobus solfataricus: Functional role in the quaternary structure thermal stabilization.

Biochemical, crystallographic, and computational data support the hypothesis that electrostatic interactions are among the dominant forces in stabilizing hyperthermophilic proteins. The thermostable beta-glycosidase from the hyperthermophile Sulfolobus solfataricus (Ssbeta-gly) is an interesting model system for the study of protein adaptation to high temperatures. The largest ion-pair network of Ssbeta-gly is located at the tetrameric interface of the molecule; in this paper, key residues in this region were modified by site-directed mutagenesis and the stability of the mutants was analyzed by kinetics of thermal denaturation. All mutations produced faster enzyme inactivation, suggesting that the C-terminal ionic network prevents the dissociation into monomers, which is the limiting step in the mechanism of Ssbeta-gly inactivation. Moreover, the calculated reaction order showed that the mechanism of inactivation depends on the mutation introduced, suggesting that intermediates maintaining enzymatic activity are produced during the inactivation transition of some, but not all, mutants. Molecular models of each mutant allow us to rationalize the experimental evidence and give support to the current theories on the mechanism of ion pair stabilization in proteins from hyperthermophiles.

Enzyme Stability↗
PubMed2002

DNA bending, compaction and negative supercoiling by the architectural protein Sso7d of Sulfolobus solfataricus.

Members of the Sso7d/Sac7d family are small, abundant, non-specific DNA-binding proteins of the hyperthermophilic Archaea SULFOLOBUS: Crystal structures of these proteins in complex with oligonucleotides showed that they induce changes in the helical twist and marked DNA bending. On this basis they have been suggested to play a role in organising chromatin structures in these prokaryotes, which lack histones. We report functional in vitro assays to investigate the effects of the observed Sso7d-induced structural modifications on DNA geometry and topology. We show that binding of multiple Sso7d molecules to short DNA fragments induces significant curvature and reduces the stiffness of the complex. Sso7d induces negative supercoiling of DNA molecules of any topology (relaxed, positively or negatively supercoiled) and in physiological conditions of temperature and template topology. Binding of Sso7d induces compaction of positively supercoiled and relaxed DNA molecules, but not of negatively supercoiled ones. Finally, Sso7d inhibits the positive supercoiling activity of the thermophile-specific enzyme reverse gyrase. The proposed biological relevance of these observations is that these proteins might model the behaviour of DNA in constrained chromatin environments.

Archaeal Proteins↗
PubMed2002

Molecular biology of extremophiles: recent progress on the hyperthermophilic archaeon Sulfolobus.

Extremophiles are microorganisms that flourish in habitats of extreme temperature, pH, salinity, or pressure. All extreme environments are dominated by microorganisms belonging to Archaea, the third domain of life, evolutionary distinct from Bacteria and Eucarya. Over the past few years the biology of extremophilic Archaea has stimulated a lot of interest, aimed at understanding at molecular level the adaptation to their life conditions, as well as their evolutionary relationships to other organisms. Here, we review recent insights in the molecular biology of thermoacidophilic Archaea of the genus Sulfolobus, which has been used as a model system for biochemical, structural, and genetic studies in Archaea and extremophiles in general. With the recent completion of the genome sequence of Sulfolobus solfataricus it is expected that these organisms will contribute new discoveries in the near future.

DNA Replication↗