Purification and characterization of DNA polymerase from the archaebacterium<i>Sulfolobus acidocaldarius</i>

Klimczak, Leszek J.; Grummt, Friedrich; Burger, K. J.

doi:10.1093/nar/13.14.5269

Cited by 41 publications

(27 citation statements)

References 17 publications

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“…nt, nucleotides. Except for the enzyme from S. acidocaldarius, for which conflicting results have been reported (13,21), all (unmodified) archaeal polymerases studied exhibit 3Ј-5Ј exonuclease activity (28). We have also detected a very weak 5Ј-3Ј exonuclease activity of the C. symbiosum polymerase on double-stranded DNA templates.…”

Section: Discussionmentioning

confidence: 53%

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Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum

et al. 1997

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Cenarchaeum symbiosum, an archaeon which lives in specific association with a marine sponge, belongs to a recently recognized nonthermophilic crenarchaeotal group that inhabits diverse cold and temperate environments. Nonthermophilic crenarchaeotes have not yet been obtained in laboratory culture, and so their phenotypic characteristics have been inferred solely from their ecological distribution. Here we report on the first protein to be characterized from one of these organisms. The DNA polymerase gene of C. symbiosum was identified in the vicinity of the rRNA operon on a large genomic contig. Its deduced amino acid sequence is highly similar to those of the archaeal family B (␣-type) DNA polymerases. It shared highest overall sequence similarity with the crenarchaeal DNA polymerases from the extreme thermophiles Sulfolobus acidocaldarius and Pyrodictium occultum (54% and 53%, respectively). The conserved motifs of B (␣-)-type DNA polymerases and 3-5 exonuclease were identified in the 845-amino-acid sequence. The 96-kDa protein was expressed in Escherichia coli and purified with affinity tags. It exhibited its highest specific activity with gapped-duplex (activated) DNA as the substrate. Single-strand-and double-strand-dependent 3-5 exonuclease activity was detected, as was a marginal 5-3 exonuclease activity. The enzyme was rapidly inactivated at temperatures higher than 40°C, with a half-life of 10 min at 46°C. It was found to be less thermostable than polymerase I of E. coli and is substantially more heat labile than its most closely related homologs from thermophilic and hyperthermophilic crenarchaeotes. Although phylogenetic studies suggest a thermophilic ancestry for C. symbiosum and its relatives, our biochemical analysis of the DNA polymerase is consistent with the postulated nonthermophilic phenotype of these crenarchaeotes, to date inferred solely from their ecological distribution.The archaeon Cenarchaeum symbiosum has recently been detected by molecular biological techniques within the tissues of the marine sponge Axinella mexicana (32). Sequence analysis of its 16S ribosomal DNA (rDNA) has revealed that C. symbiosum is specifically related to a group of crenarchaeotes that have been detected by molecular phylogenetic surveys in a number of cold marine environments, including polar seas (9,10,16,23,25), and more recently also in terrestrial soils (2,19,37) and freshwater lakes (17,24,33). C. symbiosum has been maintained in stable association with its host under controlled laboratory conditions, demonstrating that the organism thrives and reproduces at temperatures as low as 10°C (32). Its growth temperature is therefore at least 60°C below the optimum of any cultivated species of Crenarchaeota, including the extreme thermophiles of the orders Sulfolobales and Thermoproteales.Although their wide distribution and abundance in various habitats suggest a global ecological and biogeochemical significance (11, 27), virtually nothing is known to date about the phenotypic and metabolic properties of nonth...

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Section: Discussionmentioning

confidence: 53%

“…2). The protein reported here is most closely related to this second, crenarchaeotal group, e.g., to the polymerases from Sulfolobus acidocaldarius and S. solfataricus that have been purified as the major polymerization activity from these thermophiles (13,21) and to one of the two polymerases of P. occultum (38).…”

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confidence: 85%

Characterization of a DNA polymerase from the uncultivated psychrophilic archaeon Cenarchaeum symbiosum

et al. 1997

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“…The most common indicator of the presence of a eukaryotic-like DNA polymerase was sensitivity to aphidicolin, a fungal metabolite which inhibits eukaryotic DNA replication by allosterically binding to the replicative DNA polymerases (45). By using aphidicolin sensitivity as an indicator, S. acidocaldarius and S. solfataricus were found to possess a eukaryoticlike DNA polymerase activity as well as an unclassified aphidicolin-resistant DNA polymerase activity (14,29,35,41,49). Sequencing of the gene corresponding to the aphidicolin-sensitive activity from S. solfataricus MT4 confirmed that this DNA polymerase was a family B homolog more similar to eukaryotic than to eubacterial homologs (38).…”

Section: Discussionmentioning

confidence: 99%

“…Aphidicolin-sensitive DNA polymerases were subsequently purified from halophilic, methanogenic, and some thermophilic archaea, suggesting that, like eukaryotes and unlike eubacteria, archaea use an aphidicolin-sensitive DNA polymerase for DNA replication (summarized in reference 20). However, not all archaea demonstrate a sensitivity to aphidicolin, and aphidicolin-resistant DNA polymerases have recently been isolated and biochemically characterized from archaea that had been shown to also contain aphidicolin-sensitive DNA enzymes (14,25,29,35,48,49). Sequencing of the genes for several aphidicolin-sensitive and one aphidicolin-resistant DNA polymerase revealed that these DNA polymerases are all homologous to family B DNA polymerases from eubacteria and eukaryotes (34,38,48,49) and more similar at the amino acid level to eukaryotic homologs.…”

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confidence: 99%

Gene duplications in evolution of archaeal family B DNA polymerases

1997

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All archaeal DNA-dependent DNA polymerases sequenced to date are homologous to family B DNA polymerases from eukaryotes and eubacteria. Presently, representatives of the euryarchaeote division of archaea appear to have a single family B DNA polymerase, whereas two crenarchaeotes, Pyrodictium occultum and Sulfolobus solfataricus, each possess two family B DNA polymerases. We have found the gene for yet a third family B DNA polymerase, designated B3, in the crenarchaeote S. solfataricus P2. The encoded protein is highly divergent at the amino acid level from the previously characterized family B polymerases in S. solfataricus P2 and contains a number of nonconserved amino acid substitutions in catalytic domains. We have cloned and sequenced the ortholog of this gene from the closely related Sulfolobus shibatae. It is also highly divergent from other archaeal family B DNA polymerases and, surprisingly, from the S. solfataricus B3 ortholog. Phylogenetic analysis using all available archaeal family B DNA polymerases suggests that the S. solfataricus P2 B3 and S. shibatae B3 paralogs are related to one of the two DNA polymerases of P. occultum. These sequences are members of a group which includes all euryarchaeote family B homologs, while the remaining crenarchaeote sequences form another distinct group. Archaeal family B DNA polymerases together constitute a monophyletic subfamily whose evolution has been characterized by a number of gene duplication events.Studies on the mechanisms of DNA replication in eubacteria and eukaryotes have led to the identification of numerous DNA-dependent DNA polymerases (31). These have been classified into families based on amino acid sequence similarity of the catalytic subunit to one of the three Escherichia coli DNA polymerases (5). Family A DNA polymerases include E. coli DNA polymerase I (polI), all eubacterial polI homologs, some eubacterial phage DNA polymerases, and mitochondrial DNA polymerases (often called ␥ polymerase). Family B DNA polymerases include E. coli DNA polymerase II, some eubacterial phage DNA polymerases, the eukaryotic nuclear replicative DNA polymerases (␣, ␦, and ε), and eukaryotic viral and plasmid-borne enzymes. Family C includes only eubacterial polIII homologs: there are no known phage, viral, archaeal, or eukaryotic family C DNA polymerases. An additional eukaryotic nuclear encoded DNA polymerase, ␤, which functions in repair, is assigned to family X. Members of family X have little amino acid sequence similarity with DNA polymerases but instead exhibit amino acid sequence similarity to terminal transferases.Although our understanding of DNA replication in eubacteria and eukaryotes is quite advanced, comparatively little is known about DNA replication in archaea. Early studies on DNA replication showed that aphidicolin, a specific inhibitor of eukaryotic DNA replication, inhibited cell growth and DNA synthesis in halophilic archaea (21, 43). Aphidicolin-sensitive DNA polymerases were subsequently purified from halophilic, methanogenic, and some thermo...

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“…Three enzymes, PolB1, PolB2 and PolB3, belong to family B of DNA polymerases (She et al, 2001b). The replicative polymerase B1 has been most intensively studied (Klimczak et al, 1985;Rossi et al, 1986;Bukhrashvili et al, 1989;Chinchaladze et al, 1989;Elie et al, 1989; reviewed by Böhlke et al, 2002). Sulfolobus possesses also a DNA polymerase belonging to the Y family, which apparently is specialized in the replication of aberrant DNA templates and is able to bypass certain DNA lesions in an error-free manner (Kulaeva et al, 1996;Boudsocq et al, 2001;Gruz et al, 2001;Kokoshka et al, 2002;Potapova et al, 2002).…”

Section: Uridine-5′-monophosphatementioning

confidence: 99%