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Jeffrey H Miller - One of the best experts on this subject based on the ideXlab platform.
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direct interaction between uracil dna glycosylase and a proliferating cell nuclear antigen homolog in the crenarchaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2002Co-Authors: Hanjing Yang, Alessandro A. Sartori, Sorel Fitzgibbon, Malgorzata M Slupska, Juhuei Chiang, Michel Lebel, Joseph Jiricny, Jeffrey H MillerAbstract:Abstract Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp on duplex DNA. Its homologs, present in Eukarya and Archaea, are part of protein complexes that are indispensable for DNA replication and DNA repair. In Eukarya, PCNA is known to interact with more than a dozen different proteins, including a human major nuclear uracil-DNA glycosylase (hUNG2) involved in immediate postreplicative repair. In Archaea, only three classes of PCNA-binding proteins have been reported previously: replication factor C (the PCNA clamp loader), family B DNA polymerase, and flap endonuclease. In this study, we report a direct interaction between a uracil-DNA glycosylase (Pa-UDGa) and a PCNA homolog (Pa-PCNA1), both from the hyperthermophilic crenarchaeonPyrobaculum aerophilum (Topt = 100 °C). We demonstrate that the Pa-UDGa-Pa-PCNA1 complex is thermostable, and two hydrophobic amino acid residues onPa-UDGa (Phe191 and Leu192) are shown to be crucial for this interaction. It is interesting to note that although Pa-UDGa has homologs throughout the Archaea and bacteria, it does not share significant sequence similarity with hUNG2. Nevertheless, our results raise the possibility thatPa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil.
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A novel uracil‐DNA glycosylase with broad substrate specificity and an unusual active site
The EMBO Journal, 2002Co-Authors: Alessandro A. Sartori, Jeffrey H Miller, Hanjing Yang, Sorel Fitz-gibbon, Josef JiricnyAbstract:Uracil-DNA glycosylases (UDGs) catalyse the removal of uracil by flipping it out of the double helix into their binding pockets, where the glycosidic bond is hydrolysed by a water molecule activated by a polar amino acid. Interestingly, the four known UDG families differ in their active site make-up. The activating residues in UNG and SMUG enzymes are aspartates, thermostable UDGs resemble UNG-type enzymes, but carry glutamate rather than aspartate residues in their active sites, and the less active MUG/TDG enzymes contain an active site asparagine. We now describe the first member of a fifth UDG family, Pa-UDGb from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum, the active site of which lacks the polar residue that was hitherto thought to be essential for catalysis. Moreover, Pa-UDGb is the first member of the UDG family that efficiently catalyses the removal of an aberrant purine, hypoxanthine, from DNA. We postulate that this enzyme has evolved to counteract the mutagenic threat of cytosine and adenine deamination, which becomes particularly acute in organisms living at elevated temperatures.
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genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sorel Fitzgibbon, Karl O Stetter, Melvin I Simon, Heidi Ladner, Jeffrey H MillerAbstract:We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (T-opt = 100 degrees C) crenarchaeon. Clues were found suggesting explanations of the organism's surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5' untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine-Dalgarno)-based mechanism for translation initiation at the 5' end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a "mutator" phenotype.
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biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2001Co-Authors: Alessandro A. Sartori, Sorel Fitzgibbon, Jeffrey H Miller, Primo Schar, Josef JiricnyAbstract:Abstract Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G·U and G·T mispairs, respectively, that must be restored to G·C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G·C → A·T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G·U and G·T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G·U mispairs, in A·U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a ∼22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinantPa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus andThermotoga maritima. Interestingly, neitherPa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
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leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum
Journal of Molecular Biology, 2001Co-Authors: Malgorzata M Slupska, Sorel Fitzgibbon, Angela G King, John Besemer, Mark Borodovsky, Jeffrey H MillerAbstract:Abstract We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S 1 nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from −50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the −1 position and a BRE element with more frequent A bases at position −29 to −32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.
Karl O Stetter - One of the best experts on this subject based on the ideXlab platform.
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genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sorel Fitzgibbon, Karl O Stetter, Melvin I Simon, Heidi Ladner, Jeffrey H MillerAbstract:We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (T-opt = 100 degrees C) crenarchaeon. Clues were found suggesting explanations of the organism's surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5' untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine-Dalgarno)-based mechanism for translation initiation at the 5' end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a "mutator" phenotype.
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Cultivation of hyperthermophilic archaea in capillary tubes resulting in improved preservation of fine structures
Archives of Microbiology, 1997Co-Authors: Gertraud Rieger, Karl O Stetter, Karin Müller, René Hermann, Reinhard RachelAbstract:A method for cultivating hyperthermophilic archaea that results in very high cell densities and in improved structural preservation of the cells is described. Cellulose capillary tubes, originally introduced as containers for embedding for electron microscopy, were filled with cells, closed at both ends, and put into sterile culture medium. Within these capillaries, which serve as ultrafiltration chambers, cells could be cultivated to much higher cell densities than in regular cultures. The capillaries containing cells were processed for ultrathin-sectioning by fixation, freeze-substitution, and embedding. Using this cultivation procedure, centrifugation, which may destroy sensitive structural components, could be avoided, and the cells of hyperthermophilic archaea were well-preserved. These undisturbed cells revealed the following new structural features: (1) a high number of tubules in ultrathin-sections, indicating a well-preserved network of Pyrodictium cells and tubules; (2) “ultraflat areas” of Pyrodictium cells, with the two membranes being in direct contact and, at some places, bulging out, forming evaginations; (3) novel cell-to-cell connections between Thermoproteus cells and, similarly, between Pyrobaculum cells; and (4) a surface coat on Pyrobaculum aerophilum cells. The cultivation procedure offers distinct advantages over conventional techniques and might be applicable for improved electron microscopy of other sensitive microorganisms.
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Organic solutes in hyperthermophilic archaea.
Applied and Environmental Microbiology, 1997Co-Authors: Lígia O. Martins, Robert Huber, Karl O Stetter, Harald Huber, M S Da Costa, Helena SantosAbstract:We examined the accumulation of organic solutes under optimum growth conditions in 12 species of thermophilic and hyperthermophilic Archaea belonging to the Crenarchaeota and Euryarchaeota. Pyrobaculum aerophilum, Thermoproteus tenax, Thermoplasma acidophilum, and members of the order Sulfolobales accumulated trehalose. Pyrococcus furiosus accumulated di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate and (beta)-mannosylglycerate, Methanothermus fervidus accumulated cyclic-2,3-bisphosphoglycerate and (beta)-mannosylglycerate, while the only solute detected in Pyrodictium occultum was di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate. Methanopyrus kandleri accumulated large concentrations of cyclic-2,3-bisphosphoglycerate. On the other hand, Archaeoglobus fulgidus accumulated three phosphorylated solutes; prominent among them was a compound identified as di-glycerol-phosphate. This solute increased in concentration as the salinity of the medium and the growth temperature were raised, suggesting that this compound serves as a general stress solute. Di-myo-inositol-1,1(prm1)(3,3(prm1))-phosphate accumulated at supraoptimal temperature only. The relationship between the accumulation of unusual solutes and high temperatures is also discussed.
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a fosmid based genomic map and identification of 474 genes of the hyperthermophilic archaeon Pyrobaculum aerophilum
Extremophiles, 1997Co-Authors: Sorel Fitzgibbon, Karl O Stetter, Anthony J Choi, Jeffrey H Miller, Melvin I Simon, Ronald V SwansonAbstract:We have constructed a physical map of the approximately 1.7-Mb genome of the hyperthermophilic archaeon Pyrobaculum aerophilum. Derived from a 12× coverage genomic fosmid library with an average insert size of 36 Kb, the map consists of a single circular contig of 96 overlapping fosmid clones with 211 markers ordered along them. One hundred of the sequence markers have strong similarities to known genes. Many overlaps were also checked using restriction fingerprint analysis. This map is an important step in the elucidation of the sequence of the entire genome of Pyrobaculum aerophilum. To this end we have determined more than 95% of the genome with 15000 random sequences. Each sequence has been screened against the public sequence databases to identify similarities to known genes. We report here a list of the 474 putative genes we have identified.
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genomic and cdna sequence tags of the hyperthermophilic archaeon Pyrobaculum aerophilum
Nucleic Acids Research, 1996Co-Authors: Paul Volkl, Karl O Stetter, Sorel Fitzgibbon, Peter Markiewicz, Claudia Baikalov, Jeffrey H MillerAbstract:The hyperthermophilic archaeum, Pyrobaculum aerophilum, grows optimally at 100 degrees C with a doubling time of 180 min. It is a member of the phylogenetically ancient Thermoproteales order, but differs significantly from all other members by its facultatively aerobic metabolism. Due to its simple cultivation requirements and its nearly 100% plating efficiency, it was chosen as a model organism for studying the genome organization of hyperthermophilic ancient archaea. By a G+C content of the DNA of 52 mol%, sequence analysis was easily possible. At least some of the mRNA of P. aerophilum carried poly-A tails facilitating the construction of a cDNA library. 245 sequence tags of a poly-A primed cDNA library and 55 sequence tags from a 1-2 kb Sau3AI-fragment containing genomic library were analyzed and the corresponding amino acid sequences compared with protein sequences from databases. Fourteen percent of the cDNA and >9% of genomic DNA sequence tags revealed significant similarities to proteins in the databases. Matches were obtained to proteins from archaeal, bacterial and eukaryal sources. Some sequences showed greatest similarity to eukaryal rather than to bacterial versions of proteins, other matches were found to proteins which had previously only been found in eukaryotes.
Paul Volkl - One of the best experts on this subject based on the ideXlab platform.
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genomic and cdna sequence tags of the hyperthermophilic archaeon Pyrobaculum aerophilum
Nucleic Acids Research, 1996Co-Authors: Paul Volkl, Karl O Stetter, Sorel Fitzgibbon, Peter Markiewicz, Claudia Baikalov, Jeffrey H MillerAbstract:The hyperthermophilic archaeum, Pyrobaculum aerophilum, grows optimally at 100 degrees C with a doubling time of 180 min. It is a member of the phylogenetically ancient Thermoproteales order, but differs significantly from all other members by its facultatively aerobic metabolism. Due to its simple cultivation requirements and its nearly 100% plating efficiency, it was chosen as a model organism for studying the genome organization of hyperthermophilic ancient archaea. By a G+C content of the DNA of 52 mol%, sequence analysis was easily possible. At least some of the mRNA of P. aerophilum carried poly-A tails facilitating the construction of a cDNA library. 245 sequence tags of a poly-A primed cDNA library and 55 sequence tags from a 1-2 kb Sau3AI-fragment containing genomic library were analyzed and the corresponding amino acid sequences compared with protein sequences from databases. Fourteen percent of the cDNA and >9% of genomic DNA sequence tags revealed significant similarities to proteins in the databases. Matches were obtained to proteins from archaeal, bacterial and eukaryal sources. Some sequences showed greatest similarity to eukaryal rather than to bacterial versions of proteins, other matches were found to proteins which had previously only been found in eukaryotes.
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the sequence of a subtilisin type protease aerolysin from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability
Protein Science, 1994Co-Authors: Paul Volkl, Karl O Stetter, Peter Markiewicz, Jeffrey H MillerAbstract:The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 °C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80–130 °C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named “aerolysin”) was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases. Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.
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The sequence of a subtilisin‐type protease (aerolysin) from the hyperthermophilic archaeum Pyrobaculum aerophilum reveals sites important to thermostability
Protein Science, 1994Co-Authors: Paul Volkl, Karl O Stetter, Peter Markiewicz, Jeffrey H MillerAbstract:The hyperthermophilic archaeum Pyrobaculum aerophilum grows optimally at 100 °C and pH 7.0. Cell homogenates exhibit strong proteolytic activity within a temperature range of 80–130 °C. During an analysis of cDNA and genomic sequence tags, a genomic clone was recovered showing strong sequence homology to alkaline subtilisins of Bacillus sp. The total DNA sequence of the gene encoding the protease (named “aerolysin”) was determined. Multiple sequence alignment with 15 different serine-type proteases showed greatest homology with subtilisins from gram-positive bacteria rather than archaeal or eukaryal serine proteases. Models of secondary and tertiary structure based on sequence alignments and the tertiary structures of subtilisin Carlsberg, BPN', thermitase, and protease K were generated for P. aerophilum subtilisin. This allowed identification of sites potentially contributing to the thermostability of the protein. One common transition put alanines at the beginning and end of surface alpha-helices. Aspartic acids were found at the N-terminus of several surface helices, possibly increasing stability by interacting with the helix dipole. Several of the substitutions in regions expected to form surface loops were adjacent to each other in the tertiary structure model.
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Pyrobaculum aerophilum sp nov a novel nitrate reducing hyperthermophilic archaeum
Applied and Environmental Microbiology, 1993Co-Authors: Paul Volkl, Robert Huber, Elisabeth Drobner, Reinhard Rachel, Siegfried Burggraf, Antonio Trincone, Karl O StetterAbstract:A novel rod-shaped hyperthermophilic archaeum has been isolated from a boiling marine water hole at Maronti Beach, Ischia, Italy. It grew optimally at 100 degrees C and pH 7.0 by aerobic respiration as well as by dissimilatory nitrate reduction, forming dinitrogen as a final product. Organic and inorganic compounds served as substrates during aerobic and anaerobic respiration. Growth was inhibited by elemental sulfur. The cell wall was composed of a surface layer of hexameric protein complexes arranged on a p6 lattice. The core lipids consisted mainly of glycerol diphytanyl glycerol tetraethers with various degrees of cyclization. The G+C content was 52 mol%. The new isolate resembled members of the genera Thermoproteus and Pyrobaculum by its ability to form characteristic terminal spherical bodies ("golf clubs"). On the basis of its 16S rRNA sequence, the new isolate exhibited a close relationship to the genus Pyrobaculum. It is described as a new species, which we name Pyrobaculum aerophilum (type strain: IM2; DSM 7523).
David Eisenberg - One of the best experts on this subject based on the ideXlab platform.
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structure of superoxide dismutase from Pyrobaculum aerophilum presents a challenging case in molecular replacement with multiple molecules pseudo symmetry and twinning
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Michael R Sawaya, David EisenbergAbstract:: The crystal structure of superoxide dismutase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was determined by molecular replacement at 1.8 A resolution. The structure determination was made especially challenging by the large number of molecules (24) in the asymmetric unit, the presence of a pseudo-crystallographic twofold operator close to a twinning operator and the inability to detect twinning by conventional means. Molecular replacement proceeded at low resolution in pseudo (apparent) space group P3(2)12 and was facilitated by examination of the self-rotation function and native Patterson map. Refinement, however, stalled at an R factor of 40% when high-resolution data were included. Expanding to the lower symmetry space group P3(2) decreased R (to 22%) and R(free) (to 26%), but not by as much as expected for the quality of data. Finally, despite the apparent lack of evidence from conventional twinning tests [i.e. plots of the second moment of I and N(Z) distributions], a twinning operator was included in the refinement, lowering R and R(free) to 16.2 and 21.7%, respectively. The early detection of twinning appears to have been masked by a deviation in the expected intensity distribution caused by the presence of non-crystallographic translational symmetry. These findings suggest the importance of testing twinning operators in cases where pseudo-translational symmetry can explain negative results from conventional twinning tests. The structure reveals a tetrameric assembly with 222 symmetry, similar to superoxide dismutase structures from other organisms. The current structural model represents the metal-free state of the enzyme.
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Structure of superoxide dismutase from Pyrobaculum aerophilum presents a challenging case in molecular replacement with multiple molecules, pseudo‐symmetry and twinning
Acta Crystallographica Section D-biological Crystallography, 2003Co-Authors: Michael R Sawaya, David EisenbergAbstract:: The crystal structure of superoxide dismutase from the hyperthermophilic crenarchaeon Pyrobaculum aerophilum was determined by molecular replacement at 1.8 A resolution. The structure determination was made especially challenging by the large number of molecules (24) in the asymmetric unit, the presence of a pseudo-crystallographic twofold operator close to a twinning operator and the inability to detect twinning by conventional means. Molecular replacement proceeded at low resolution in pseudo (apparent) space group P3(2)12 and was facilitated by examination of the self-rotation function and native Patterson map. Refinement, however, stalled at an R factor of 40% when high-resolution data were included. Expanding to the lower symmetry space group P3(2) decreased R (to 22%) and R(free) (to 26%), but not by as much as expected for the quality of data. Finally, despite the apparent lack of evidence from conventional twinning tests [i.e. plots of the second moment of I and N(Z) distributions], a twinning operator was included in the refinement, lowering R and R(free) to 16.2 and 21.7%, respectively. The early detection of twinning appears to have been masked by a deviation in the expected intensity distribution caused by the presence of non-crystallographic translational symmetry. These findings suggest the importance of testing twinning operators in cases where pseudo-translational symmetry can explain negative results from conventional twinning tests. The structure reveals a tetrameric assembly with 222 symmetry, similar to superoxide dismutase structures from other organisms. The current structural model represents the metal-free state of the enzyme.
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Genomic evidence that the intracellular proteins of archaeal microbes contain disulfide bonds
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Parag Mallick, David Eisenberg, Daniel R. Boutz, Todd O YeatesAbstract:Disulfide bonds have only rarely been found in intracellular proteins. That pattern is consistent with the chemically reducing environment inside the cells of well-studied organisms. However, recent experiments and new calculations based on genomic data of archaea provide striking contradictions to this pattern. Our results indicate that the intracellular proteins of certain hyperthermophilic archaea, especially the crenarchaea Pyrobaculum aerophilum and Aeropyrum pernix, are rich in disulfide bonds. This finding implicates disulfide bonding in stabilizing many thermostable proteins and points to novel chemical environments inside these microbes. These unexpected results illustrate the wealth of biochemical insights available from the growing reservoir of genomic data.
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structure of a nudix protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit beta sheets
Acta Crystallographica Section D-biological Crystallography, 2002Co-Authors: Shuishu Wang, Cameron Mura, Michael R Sawaya, Duilio Cascio, David EisenbergAbstract:Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the β-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved β-sheet of 11 β-strands and two α-helices, forming an α–β–α sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first α-helix and the β-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein.
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Structure of a Nudix protein from Pyrobaculum aerophilum reveals a dimer with two intersubunit beta-sheets.
Acta crystallographica. Section D Biological crystallography, 2002Co-Authors: Shuishu Wang, Cameron Mura, Michael R Sawaya, Duilio Cascio, David EisenbergAbstract:Nudix proteins, formerly called MutT homolog proteins, are a large family of proteins that play an important role in reducing the accumulation of potentially toxic compounds inside the cell. They hydrolyze a wide variety of substrates that are mainly composed of a nucleoside diphosphate linked to some other moiety X and thus are called Nudix hydrolases. Here, the crystal structure of a Nudix hydrolase from the hyperthermophilic archaeon Pyrobaculum aerophilum is reported. The structure was determined by the single-wavelength anomalous scattering method with data collected at the peak anomalous wavelength of an iridium-derivatized crystal. It reveals an extensive dimer interface, with each subunit contributing two strands to the beta-sheet of the other subunit. Individual subunits consist of a mixed highly twisted and curved beta-sheet of 11 beta-strands and two alpha-helices, forming an alpha-beta-alpha sandwich. The conserved Nudix box signature motif, which contains the essential catalytic residues, is located at the first alpha-helix and the beta-strand and loop preceding it. The unusually short connections between secondary-structural elements, together with the dimer form of the structure, are likely to contribute to the thermostability of the P. aerophilum Nudix protein.
Sorel Fitzgibbon - One of the best experts on this subject based on the ideXlab platform.
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direct interaction between uracil dna glycosylase and a proliferating cell nuclear antigen homolog in the crenarchaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2002Co-Authors: Hanjing Yang, Alessandro A. Sartori, Sorel Fitzgibbon, Malgorzata M Slupska, Juhuei Chiang, Michel Lebel, Joseph Jiricny, Jeffrey H MillerAbstract:Abstract Proliferating cell nuclear antigen (PCNA) acts as a sliding clamp on duplex DNA. Its homologs, present in Eukarya and Archaea, are part of protein complexes that are indispensable for DNA replication and DNA repair. In Eukarya, PCNA is known to interact with more than a dozen different proteins, including a human major nuclear uracil-DNA glycosylase (hUNG2) involved in immediate postreplicative repair. In Archaea, only three classes of PCNA-binding proteins have been reported previously: replication factor C (the PCNA clamp loader), family B DNA polymerase, and flap endonuclease. In this study, we report a direct interaction between a uracil-DNA glycosylase (Pa-UDGa) and a PCNA homolog (Pa-PCNA1), both from the hyperthermophilic crenarchaeonPyrobaculum aerophilum (Topt = 100 °C). We demonstrate that the Pa-UDGa-Pa-PCNA1 complex is thermostable, and two hydrophobic amino acid residues onPa-UDGa (Phe191 and Leu192) are shown to be crucial for this interaction. It is interesting to note that although Pa-UDGa has homologs throughout the Archaea and bacteria, it does not share significant sequence similarity with hUNG2. Nevertheless, our results raise the possibility thatPa-UDGa may be a functional analog of hUNG2 for PCNA-dependent postreplicative removal of misincorporated uracil.
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genome sequence of the hyperthermophilic crenarchaeon Pyrobaculum aerophilum
Proceedings of the National Academy of Sciences of the United States of America, 2002Co-Authors: Sorel Fitzgibbon, Karl O Stetter, Melvin I Simon, Heidi Ladner, Jeffrey H MillerAbstract:We determined and annotated the complete 2.2-megabase genome sequence of Pyrobaculum aerophilum, a facultatively aerobic nitrate-reducing hyperthermophilic (T-opt = 100 degrees C) crenarchaeon. Clues were found suggesting explanations of the organism's surprising intolerance to sulfur, which may aid in the development of methods for genetic studies of the organism. Many interesting features worthy of further genetic studies were revealed. Whole genome computational analysis confirmed experiments showing that P. aerophilum (and perhaps all crenarchaea) lack 5' untranslated regions in their mRNAs and thus appear not to use a ribosome-binding site (Shine-Dalgarno)-based mechanism for translation initiation at the 5' end of transcripts. Inspection of the lengths and distribution of mononucleotide repeat-tracts revealed some interesting features. For instance, it was seen that mononucleotide repeat-tracts of Gs (or Cs) are highly unstable, a pattern expected for an organism deficient in mismatch repair. This result, together with an independent study on mutation rates, suggests a "mutator" phenotype.
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biochemical characterization of uracil processing activities in the hyperthermophilic archaeon Pyrobaculum aerophilum
Journal of Biological Chemistry, 2001Co-Authors: Alessandro A. Sartori, Sorel Fitzgibbon, Jeffrey H Miller, Primo Schar, Josef JiricnyAbstract:Abstract Deamination of cytosine to uracil and 5-methylcytosine to thymine represents a major mutagenic threat particularly at high temperatures. In double-stranded DNA, these spontaneous hydrolytic reactions give rise to G·U and G·T mispairs, respectively, that must be restored to G·C pairs prior to the next round of DNA replication; if left unrepaired, 50% of progeny DNA would acquire G·C → A·T transition mutations. The genome of the hyperthermophilic archaeon Pyrobaculum aerophilum has been recently shown to encode a protein, Pa-MIG, a member of the endonuclease III family, capable of processing both G·U and G·T mispairs. We now show that this latter activity is undetectable in crude extracts of P. aerophilum. However, uracil residues in G·U mispairs, in A·U pairs, and in single-stranded DNA were efficiently removed in these extracts. These activities were assigned to a ∼22-kDa polypeptide named Pa-UDG (P. aerophilum uracil-DNA glycosylase). The recombinantPa-UDG protein is highly thermostable and displays a considerable degree of homology to the recently described uracil-DNA glycosylases from Archaeoglobus fulgidus andThermotoga maritima. Interestingly, neitherPa-MIG nor Pa-UDG was inhibited by UGI, a generic inhibitor of the UNG family of uracil glycosylases. Yet a small fraction of the total uracil processing activity present in crude extracts of P. aerophilum was inhibited by this peptide. This implies that the hyperthermophilic archaeon possesses at least a three-pronged defense against the mutagenic threat of hydrolytic deamination of cytosines in its genomic DNA.
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leaderless transcripts of the crenarchaeal hyperthermophile Pyrobaculum aerophilum
Journal of Molecular Biology, 2001Co-Authors: Malgorzata M Slupska, Sorel Fitzgibbon, Angela G King, John Besemer, Mark Borodovsky, Jeffrey H MillerAbstract:Abstract We mapped transcription start sites for ten unrelated protein-encoding Pyrobaculum aerophilum genes by primer extension and S 1 nuclease mapping. All of the mapped transcripts start at the computationally predicted translation start codons, two of which were supported by N-terminal protein sequencing. A whole genome computational analysis of the regions from −50 to +50 nt around the predicted translation starts codons revealed a clear upstream pattern matching the consensus sequence of the archaeal TATA box located unusually close to the translation starts. For genes with the TATA boxes that best matched the consensus sequence, the distance between the TATA box and the translation start codon appears to be shorter than 30 nt. Two other promoter elements distinguished were also found unusually close to the translation start codons: a transcription initiator element with significant elevation of C and T frequencies at the −1 position and a BRE element with more frequent A bases at position −29 to −32 (counting from the translation start site). We also show that one of the mapped genes is transcribed as the first gene of an operon. For a set of genes likely to be internal in operons the upstream signal extracted by computer analysis was a Shine-Dalgarno pattern matching the complementary sequence of P. aerophilum 16 S rRNA. Together these results suggest that the translation of proteins encoded by single genes or genes that are first in operons in the hyperthermophilic crenarchaeon P. aerophilum proceeds mostly, if not exclusively, through leaderless transcripts. Internal genes in operons are likely to undergo translation via a mechanism that is facilitated by ribosome binding to the Shine-Dalgarno sequence.
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a thermostable endonuclease iii homolog from the archaeon Pyrobaculum aerophilum
Nucleic Acids Research, 2001Co-Authors: Hanjing Yang, Sorel Fitzgibbon, Isabella T Phan, Mahmud K K Shivji, Richard D Wood, Wendy M Clendenin, Elizabeth C Hyman, Jeffrey H MillerAbstract:: Pyrimidine adducts in cellular DNA arise from modification of the pyrimidine 5,6-double bond by oxidation, reduction or hydration. The biological outcome includes increased mutation rate and potential lethality. A major DNA N:-glycosylase responsible for the excision of modified pyrimidine bases is the base excision repair (BER) glycosylase endonuclease III, for which functional homologs have been identified and characterized in Escherichia coli, yeast and humans. So far, little is known about how hyperthermophilic Archaea cope with such pyrimidine damage. Here we report characterization of an endonuclease III homolog, PaNth, from the hyperthermophilic archaeon Pyrobaculum aerophilum, whose optimal growth temperature is 100 degrees C. The predicted product of 223 amino acids shares significant sequence homology with several [4Fe-4S]-containing DNA N:-glycosylases including E.coli endonuclease III (EcNth). The histidine-tagged recombinant protein was expressed in E.coli and purified. Under optimal conditions of 80-160 mM NaCl and 70 degrees C, PaNth displays DNA glycosylase/ss-lyase activity with the modified pyrimidine base 5,6-dihydrothymine (DHT). This activity is enhanced when DHT is paired with G. Our data, showing the structural and functional similarity between PaNth and EcNth, suggests that BER of modified pyrimidines may be a conserved repair mechanism in Archaea. Conserved amino acid residues are identified for five subfamilies of endonuclease III/UV endonuclease homologs clustered by phylogenetic analysis.
