Characterization of two DNA polymerases from the hyperthermophilic euryarchaeon Pyrococcus abyssi.

The complete genome sequence of the hyperthermophilic archaeon Pyrococcus abyssi revealed the presence of a family B DNA polymerase (Pol I) and a family D DNA polymerase (Pol II). To extend our knowledge about euryarchaeal DNA polymerases, we cloned the genes encoding these two enzymes and expressed them in Escherichia coli. The DNA polymerases (Pol I and Pol II) were purified to homogeneity and characterized. Pol I had a molecular mass of approximately 90 kDa, as estimated by SDS/PAGE. The optimum pH and Mg(2+) concentration of Pol I were 8.5-9.0 and 3 mm, respectively. Pol II is composed of two subunits that are encoded by two genes arranged in tandem on the P. abyssi genome. We cloned these genes and purified the Pol II DNA polymerase from an E. coli strain coexpressing the cloned genes. The optimum pH and Mg(2+) concentration of Pol II were 6.5 and 15-20 mm, respectively. Both P. abyssi Pol I and Pol II have associated 3'-->5' exonuclease activity although the exonuclease motifs usually found in DNA polymerases are absent in the archaeal family D DNA polymerase sequences. Sequence analysis has revealed that the small subunit of family D DNA polymerase and the Mre11 nucleases belong to the calcineurin-like phosphoesterase superfamily and that residues involved in catalysis and metal coordination in the Mre11 nuclease three-dimensional structure are strictly conserved in both families. One hypothesis is that the phosphoesterase domain of the small subunit is responsible for the 3'-->5' exonuclease activity of family D DNA polymerase. These results increase our understanding of euryarchaeal DNA polymerases and are of importance to push forward the complete understanding of the DNA replication in P. abyssi.     

ATP synthesis at 100 degrees C by an ATPase purified from the hyperthermophilic archaeon Pyrodictium abyssi

The chemolithoautotrophic archaeon Pyrodictium abyssi isolate TAG 11 lives close to 100 degrees C and gains energy by sulfur respiration, with hydrogen as electron donor. From the membranes of this hyperthermophile, an ATPase complex was isolated. The purified enzyme consists of six major polypeptides, the 67, 51, 41, 26 and 22 kDa subunits composing the AF(1) headpiece, and the 7 kDa proteolipid of the AF(0) component. The headpiece of the enzyme restored the formation of ATP during sulfur respiration in membrane vesicles from which it had been removed by low salt treatment. Characteristics of the reconstituted activity suggest that the same enzyme is responsible for ATP formation in untreated membranes. ATP formation was neither sensitive to ionophores and uncouplers, nor to dicyclohexyl carbodiimide, but depended on closed vesicles. Both ATPase activity (up to 2 micromol per min and mg protein) as well as ATP formation (up to 0.4 micromol per min and mg membrane protein) were highest at 100 degrees C. A P/e2 ratio of close to one can be estimated for sulfur respiration with hydrogen. In addition to ATP, autoradiographic detection revealed the formation of high quantities of (33)P(i)-labeled ADP and of another compound not identified so far.     

The hyperthermophilic euryarchaeota Pyrococcus abyssi likely requires the two DNA polymerases D and B for DNA replication

DNA polymerases carry out DNA synthesis during DNA replication, DNA recombination and DNA repair. During the past five years, the number of DNA polymerases in both eukarya and bacteria has increased to at least 19 and multiple biological roles have been assigned to many DNA polymerases. Archaea, the third domain of life, on the other hand, have only a subset of the eukaryotic-like DNA polymerases. The diversity among the archaeal DNA polymerases poses the intriguing question of their functional tasks. Here, we focus on the two identified DNA polymerases, the family B DNA polymerase B (PabpolB) and the family D DNA polymerase D (PabpolD) from the hyperthermophilic euryarchaeota Pyrococcus abyssi. Our data can be summarized as follows: (i) both Pabpols are DNA polymerizing enzymes exclusively; (ii) their DNA binding properties as tested in gel shift competition assays indicated that PabpolD has a preference for a primed template; (iii) PabPolD is a primer-directed DNA polymerase independently of the primer composition whereas PabpolB behaves as an exclusively DNA primer-directed DNA polymerase; (iv) PabPCNA is required for PabpolD to perform efficient DNA synthesis but not PabpolB; (v) PabpolD, but not PabpolB, contains strand displacement activity; (vii) in the presence of PabPCNA, however, both Pabpols D and B show strand displacement activity; and (viii) we show that the direct interaction between PabpolD and PabPCNA is DNA-dependent. Our data imply that PabPolD might play an important role in DNA replication likely together with PabpolB, suggesting that archaea require two DNA polymerases at the replication fork.     

Methylpurine DNA glycosylase of the hyperthermophilic archaeon Archaeoglobus fulgidus

Base excision repair of DNA alkylation damage is initiated by a methylpurine DNA glycosylase (MPG) function. Such enzymes have previously been characterized from bacteria and eukarya, but not from archaea. We identified activity for the release of methylated bases from DNA in cell-free extracts of Archaeoglobus fulgidus, an archaeon growing optimally at 83 degrees C. An open reading frame homologous to the alkA gene of Escherichia coli was overexpressed and identified as a gene encoding an MPG enzyme (M(r) = 34 251), hereafter designated afalkA. The purified AfalkA protein differs from E. coli AlkA by excising alkylated bases only, from DNA, in the following order of efficiency: 3-methyladenine (m(3)A) > 3-methylguanine approximately 7-methyladenine > 7-methylguanine. Although the rate of enzymatic release of m(3)A is highest in the temperature range of 65-75 degrees C, it is only reduced by 50% at 45 degrees C, a temperature that does not support growth of A. fulgidus. At temperatures above 75 degrees C, nonenzymatic release of methylpurines predominates. The results suggest that the biological function of AfalkA is to excise m(3)A from DNA at suboptimal and maybe even mesophilic temperatures. This hypothesis is further supported by the observation that the afalkA gene function suppresses the alkylation sensitivity of the E. coli tag alkA double mutant. The amino acid sequence similarity and evolutionary relationship of AfalkA with other MPG enzymes from the three domains of life are described and discussed.     

The DNA polymerases of Chinese hamster cells. Subcellular distribution and properties of two DNA polymerases.

We have fractionated homogenates of Chinese hamster cells grown in tissue culture, and found that >80% of those cells' DNA-dependent DNA polymerase appears localized in the soluble cytoplasm. The Chinese hamster cytoplasmic DNA polymerase is very similar to DNA polymerases from several mammalian sources: it is large and heterogeneous (165,000–200,000 daltons), sensitive to sulfhydryl-blocking reagents and absolutely requires double stranded templates containing free 3′-OH primers. Two distinct species of DNA polymerase also have been isolated from purified Chinese hamster nuclei. One nuclear DNA polymerase appeared to be identical to DNA polymerase found in the cells' soluble cytoplasm. The second polymerase, comprising 1.5–3% of the total DNA polymerase activity, was found only in nuclear extracts. That enzyme is resistant to sulfhydryl-blocking reagents and has an apparent molecular weight of 49,000. The data discussed in this report suggest that Chinese hamster cells, like other mammalian cell types, possess at least two DNA-dependent DNA polymerases that might participate in replicative DNA biosynthesis.     

Hydrogen-oxidizing electron transport components in the hyperthermophilic archaebacterium Pyrodictium brockii.

The hyperthermophilic archaebacterium Pyrodictium brockii grows optimally at 105 degrees C by a form of metabolism known as hydrogen-sulfur autotrophy, which is characterized by the oxidation of H2 by S0 to produce ATP and H2S. UV-irradiated membranes were not able to carry out the hydrogen-dependent reduction of sulfur. However, the activity could be restored by the addition of ubiquinone Q10 or ubiquinone Q6 to the UV-damaged membranes. A quinone with thin-layer chromatography migration properties similar to those of Q6 was purified by thin-layer chromatography from membranes of P. brockii, but nuclear magnetic resonance analysis failed to confirm its identity as a ubiquinone. P. brockii quinone was capable of restoring hydrogen-dependent sulfur reduction to UV-irradiated membranes. Hydrogen-reduced-minus-air-oxidized absorption difference spectra on membranes revealed absorption peaks characteristic of c-type cytochromes. A c-type cytochrome with alpha, beta, and gamma peaks at 553, 522, and 421 nm, respectively, was solubilized from membranes with 0.5% Triton X-100. Pyridine ferrohemochrome spectra confirmed its identity as a c-type cytochrome, and heme staining of membranes loaded on sodium dodecyl sulfate gels revealed a single heme-containing component of 13 to 14 kDa. Studies with the ubiquinone analog 2-n-heptyl-4-hydroxyquinoline-N-oxide demonstrated that the P. brockii quinone is located on the substrate side of the electron transport chain with respect to the c-type cytochrome. These first characterizations of the strictly anaerobic, presumably primitive P. brockii electron transport chain suggest that the hydrogenase operates at a relatively high redox potential and that the H2-oxidizing chain more closely resembles those of aerobic eubacterial H2-oxidizing bacteria than those of the H2-metabolizing systems of anaerobes or the hyperthermophile Pyrococcus furiosus.     

Identification and characterization of a DNA primase from the hyperthermophilic archaeon Methanococcus jannaschii.

We report the identification and characterisation of a DNA primase from the thermophilic methanogenic archaeon Methanococcus jannaschii (Mjpri). The analysis of the complete genome sequence of this organism has identified an open reading frame coding for a protein with sequence similarity to the small subunit of the eukaryotic DNA primase (the p50 subunit of the polymerase alpha-primase complex). This protein has been overexpressed in Escherichia coli and purified to near homogeneity. Recombinant Mjpri is able to synthesise oligoribonucleotides on various pyrimidine single-stranded DNA templates [poly(dT) and poly(dC)]. This activity requires divalent cations such Mg(2+), Mn(2+)or Zn(2+), and is additionally stimulated by the monovalent cation K(+). A multiple sequence alignment has revealed that most of the regions that are conserved in eukaryotic p50 subunits are also present in the archaeal primases, including the conserved negatively charged residues, which have been shown to be essential for catalysis in the mouse primase. Of the four cysteine residues that have been postulated to make up a putative Zn-binding motif, two are not present in the archaeal homologue. This is the first report on the biochemical characterisation of an archaeal DNA primase.     

A DNA ligase from a hyperthermophilic archaeon with unique cofactor specificity

A gene encoding DNA ligase (lig(Tk)) from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, has been cloned and sequenced, and its protein product has been characterized. lig(Tk) consists of 1,686 bp, corresponding to a polypeptide of 562 amino acids with a predicted molecular mass of 64,079 Da. Sequence comparison with previously reported DNA ligases and the presence of conserved motifs suggested that Lig(Tk) was an ATP-dependent DNA ligase. Phylogenetic analysis indicated that Lig(Tk) was closely related to the ATP-dependent DNA ligase from Methanobacterium thermoautotrophicum DeltaH, a moderate thermophilic archaeon, along with putative DNA ligases from Euryarchaeota and Crenarchaeota. We expressed lig(Tk) in Escherichia coli and purified the recombinant protein. Recombinant Lig(Tk) was monomeric, as is the case for other DNA ligases. The protein displayed DNA ligase activity in the presence of ATP and Mg(2+). The optimum pH of Lig(Tk) was 8.0, the optimum concentration of Mg(2+), which was indispensable for the enzyme activity, was 14 to 18 mM, and the optimum concentration of K(+) was 10 to 30 mM. Lig(Tk) did not display single-stranded DNA ligase activity. At enzyme concentrations of 200 nM, we observed significant DNA ligase activity even at 100 degrees C. Unexpectedly, Lig(Tk) displayed a relatively small, but significant, DNA ligase activity when NAD(+) was added as the cofactor. Treatment of NAD(+) with hexokinase did not affect this activity, excluding the possibility of contaminant ATP in the NAD(+) solution. This unique cofactor specificity was also supported by the observation of adenylation of Lig(Tk) with NAD(+). This is the first biochemical study of a DNA ligase from a hyperthermophilic archaeon.     

DNA polymerases BI and D from the hyperthermophilic archaeon Pyrococcus furiosus both bind to proliferating cell nuclear antigen with their C-terminal PIP-box motifs

Proliferating cell nuclear antigen (PCNA) is the sliding clamp that is essential for the high processivity of DNA synthesis during DNA replication. Pyrococcus furiosus, a hyperthermophilic archaeon, has at least two DNA polymerases, polymerase BI (PolBI) and PolD. Both of the two DNA polymerases interact with the archaeal P. furiosus PCNA (PfuPCNA) and perform processive DNA synthesis in vitro. This phenomenon, in addition to the fact that both enzymes display 3'-5' exonuclease activity, suggests that both DNA polymerases work in replication fork progression. We demonstrated here that both PolBI and PolD functionally interact with PfuPCNA at their C-terminal PIP boxes. The mutant PolBI and PolD enzymes lacking the PIP-box sequence do not respond to the PfuPCNA at all in an in vitro primer extension reaction. This is the first experimental evidence that the PIP-box motif, located at the C termini of the archaeal DNA polymerases, is actually critical for PCNA binding to form a processive DNA-synthesizing complex.     

DNA polymerases of anucleated cells. Isolation and characterization of two DNA polymerases from human platelets.

Two different DNA polymerases have been purified and characterized from human platelets. In the mitochondrial fraction a unique activity of the polymerase gamma type has been found. The same enzyme is found in the extramitochondrial supernatant. A second DNA polymerase, called 'cytoplasmic' DNA polymerase has been found in the 10000 x g supernatant of human platelets. The following properties of the latter DNA polymerase from human platelets are identical to those of DNA polymerase alpha from normal cells: DEAE-cellulose and phosphocellulose chromatography, size, thermal stability, phosphonoacetic acid and ethidium bromide inhibition. However, some of its properties, like high resistance to N-ethylmaleimide and the lack of DNA polymerization using synthetic RNA primers, are those of DNA polymerase beta.     

DNA topoisomerase III from the hyperthermophilic archaeon Sulfolobus solfataricus with specific DNA cleavage activity

We report the production, purification, and characterization of a type IA DNA topoisomerase, previously designated topoisomerase I, from the hyperthermophilic archaeon Sulfolobus solfataricus. The protein was capable of relaxing negatively supercoiled DNA at 75 degrees C in the presence of Mg2+. Mutation of the putative active site Tyr318 to Phe318 led to the inactivation of the protein. The S. solfataricus enzyme cleaved oligonucleotides in a sequence-specific fashion. The cleavage occurred only in the presence of a divalent cation, preferably Mg2+. The cofactor requirement of the enzyme was partially satisfied by Cu2+, Co2+, Mn2+, Ca2+, or Ni2+. It appears that the enzyme is active with a broader spectrum of metal cofactors in DNA cleavage than in DNA relaxation (Mg2+ and Ca2+). The enzyme-catalyzed oligonucleotide cleavage required at least 7 bases upstream and 2 bases downstream of the cleavage site. Analysis of cleavage by the S. solfataricus enzyme on a set of oligonucleotides revealed a consensus cleavage sequence of the enzyme: 5'-G(A/T)CA(T)AG(T)G(A)X / XX-3'. This sequence bears more resemblance to the preferred cleavage sites of topoisomerases III than to those of topoisomerases I. Based on these data and sequence analysis, we designate the enzyme S. solfataricus topoisomerase III.     

Extreme resistance to thermally induced DNA backbone breaks in the hyperthermophilic archaeon Pyrococcus furiosus.

Pyrococcus furiosus is a hyperthermophilic archaeon that grows optimally at 100 degrees C. It is not conceivable that these organisms could survive with genomic DNA that was subject to thermal destruction, yet the mechanisms protecting the genomes of this and other hyperthermophiles against such destruction are obscure. We have determined the effect of elevated temperatures up to 110 degrees C on the molecular weight of DNA in intact P. furiosus cells, compared with the effect of elevated temperatures on DNA in the mesothermophilic bacterium Escherichia coli. At 100 degrees C, DNA in P. furiosus cells is about 20 times more resistant to thermal breakage than that in E. coli cells, and six times fewer breaks were found in P. furiosus DNA after exposure to 110 degrees C for 30 min than in E. coli DNA at 95 degrees C. Our hypothesis for this remarkable stability of DNA in a hyperthermophile is that this hyperthermophile possesses DNA-binding proteins that protect against hydrolytic damage, as well as other endogenous protective mechanisms and DNA repair enzyme systems.     

Repair activities of 8-oxoguanine DNA glycosylase from Archaeoglobus fulgidus, a hyperthermophilic archaeon.

Oxidative DNA damage is caused by reactive oxygen species formed in cells as by products of aerobic metabolism or of oxidative stress. The 8-oxoguanine (8-oxoG) DNA glycosylase from Archaeoglobus fulgidus (Afogg), which excises an oxidatively-damaged form of guanine, was overproduced in Escherichia coli, purified and characterized. A. fulgidus is a sulfate-reducing archaeon, which grows at between 60 and 95 degrees C, with an optimum growth at 83 degrees C. The Afogg enzyme has both DNA glycosylase and apurinic/apyrimidinic (AP) lyase activities, with the latter proceeding through a Schiff base intermediate. As expected for a protein from a hyperthermophilic organism, the enzyme activity is optimal near pH 8.5 and 60 degrees C, denaturing at 80 degrees C, and is thermally stable at high levels of salt (500mM). The Afogg protein efficiently cleaves oligomers containing 8-oxoG:C and 8-oxoG:G base pairs, and is less effective on oligomers containing 8-oxoG:T and 8-oxoG:A mispairs. While the catalytic action mechanism of Afogg protein is likely similar to the human Ogg1 (hOgg1), the DNA recognition mechanism and the basis for 8-oxoG substrate specificity of Afogg differ from that of hOgg.     

Comprehensive search for DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus

DNA polymerase activities were scanned in a Pyrococcus furiosus cell extract to identify all of the DNA polymerases in this organism. Three main fractions containingDNA polymerizing activity were subjected to Western blot analyses, which revealed that the main activities in each fraction were derived from three previously identified DNA polymerases. PCNA (proliferating cell nuclear antigen), the sliding clamp of DNA polymerases, did not bind tightly to any of the three DNA polymerases. A primer usage preference was also shown for each purified DNA polymerase. Considering their biochemical properties, the roles of the three DNA polymerases during DNA replication in the cells are discussed.     

Crystal structure of DNA polymerase from hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1

The crystal structure of family B DNA polymerase from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 (KOD DNA polymerase) was determined. KOD DNA polymerase exhibits the highest known extension rate, processivity and fidelity. We carried out the structural analysis of KOD DNA polymerase in order to clarify the mechanisms of those enzymatic features. Structural comparison of DNA polymerases from hyperthermophilic archaea highlighted the conformational difference in Thumb domains. The Thumb domain of KOD DNA polymerase shows an "opened" conformation. The fingers subdomain possessed many basic residues at the side of the polymerase active site. The residues are considered to be accessible to the incoming dNTP by electrostatic interaction. A beta-hairpin motif (residues 242-249) extends from the Exonuclease (Exo) domain as seen in the editing complex of the RB69 DNA polymerase from bacteriophage RB69. Many arginine residues are located at the forked-point (the junction of the template-binding and editing clefts) of KOD DNA polymerase, suggesting that the basic environment is suitable for partitioning of the primer and template DNA duplex and for stabilizing the partially melted DNA structure in the high-temperature environments. The stabilization of the melted DNA structure at the forked-point may be correlated with the high PCR performance of KOD DNA polymerase, which is due to low error rate, high elongation rate and processivity.     

Genetic analysis of DNA repair in the hyperthermophilic archaeon, Thermococcus kodakaraensis

Extensive biochemical and structural analyses have been performed on the putative DNA repair proteins of hyperthermophilic archaea, in contrast to the few genetic analyses of the genes encoding these proteins. Accordingly, little is known about the repair pathways used by archaeal cells at high temperature. Here, we attempted to disrupt the genes encoding the potential repair proteins in the genome of the hyperthermophilic archaeon Thermococcus kodakaraensis. We succeeded in isolating null mutants of the hjc, hef, hjm, xpb, and xpd genes, but not the radA, rad50, mre11, herA, nurA, and xpg/fen1 genes. Phenotypic analyses of the gene-disrupted strains showed that the xpb and xpd null mutants are only slightly sensitive to ultraviolet (UV) irradiation, methyl methanesulfonate (MMS) and mitomycin C (MMC), as compared with the wild-type strain. The hjm null mutant showed sensitivity specifically to mitomycin C. On the other hand, the null mutants of the hjc gene lacked increasing sensitivity to any type of DNA damage. The Hef protein is particularly important for maintaining genome homeostasis, by functioning in the repair of a wide variety of DNA damage in T. kodakaraensis cells. Deletion of the entire hef gene or of the segments encoding either its nuclease or helicase domain produced similar phenotypes. The high sensitivity of the Δhef mutants to MMC suggests that Hef performs a critical function in the repair process of DNA interstrand cross-links. These damage-sensitivity profiles suggest that the archaeal DNA repair system has processes depending on repair-related proteins different from those of eukaryotic and bacterial DNA repair systems using homologous repair proteins analyzed here.     

A DNA polymerase from the archaeon Sulfolobus solfataricus shows sequence similarity to family B DNA polymerases.

The gene encoding the thermostable DNA polymerase from the archaeon Sulfolobus solfataricus (strain MT 4) was isolated by means of two degenerate oligonucleotide probes. They were designed on the basis of partial enzyme amino acid sequences. The gene was found to encode a 882 residues polypeptide chain with a deduced molecular mass of about 100 kDa. By comparison with other archaeal genes, putative regulatory sites were identified in the gene-flanking regions. By computer-assisted homology search, several sequence similarities among S. solfataricus and family B DNA polymerases were found. In addition, conserved sequence motifs, implicated in the 3'-5' exonuclease activity of E. coli DNA polymerase I and shared by various family A and B DNA polymerases, were also identified. This result suggests that the proofreading domains of all these enzymes are evolutionarily related.