A DNA polymerase from a thermoacidophilic archaebacterium: evolutionary and technological interests

The archaebacteria constitute a group of prokaryotes with an intermediate phylogenetic position between eukaryotes and eubacteria. The study of their DNA polymerases may provide valuable information about putative evolutionary relationships between prokaryotic and eukaryotic DNA polymerases. As a first step towards this goal, we have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. This enzyme is a monomeric protein of 100 kDa which can catalyze DNA synthesis using either activated calf thymus DNA or oligonucleotide-primed single-stranded DNA as a template. The activity is optimal at 70 degrees C and the enzyme is thermostable up to 80 degrees C; however, it can still polymerize up to 200 nucleotides at 100 degrees C. These remarkable thermophilic properties and thermostability permit examination of the mechanism of DNA synthesis under conditions of decreased stability of the DNA helix. Furthermore, these properties make S. acidocaldarius DNA polymerase a very efficient enzyme to be used in DNA amplification by the recently developed polymerase chain reaction method (PCR) as well as in the Sanger DNA sequencing technique.     

Purification and characterization of DNA polymerase from the archaebacterium Sulfolobus acidocaldarius.

DNA polymerase has been purified about 25,000-fold from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. On SDS-PAGE the enzyme was observed to have a molecular weight of 100 kDa and to be about 90% pure. The native molecular weight was 108 kDa indicating that the enzyme is composed of a single polypeptide. Activity gel analysis showed an active polypeptide of about 100 kDa. Under conditions promoting proteolysis this polypeptide was degraded to a slightly smaller form of 98 kDa. The enzyme has been characterized in respect to optimal assay conditions, template specificity, sensitivity to inhibitors and associated nuclease activities. The high temperature optimum of 65 degrees C should be emphasized. No substantial similarities have been found with other prokaryotic and eukaryotic DNA polymerases, although the enzyme bears certain resemblances to prokaryotic non-replicative polymerases.     

ATP-dependent DNA topoisomerase from the archaebacterium Sulfolobus acidocaldarius. Relaxation of supercoiled DNA at high temperature

A topoisomerase, able to relax negatively supercoiled DNA, has been isolated from the archaebacterium Sulfolobus acidocaldarius. Relaxation was fully efficient in vitro between 70 degrees C and 80 degrees C and was dependent on the presence of ATP and magnesium ions. The enzyme did not exhibit gyrase-like activity and was poorly sensitive to gyrase inhibitors. These properties are reminiscent of eukaryotic type II topoisomerases. However, the enzyme was unable to relax positively supercoiled DNA. This thermophilic enzyme may be used in a variety of ways to study the structure and stability of DNA at high temperature.     

Meiosis in Coprinus: characterization and activities of two forms of DNA polymerase during meiotic stages.

Two forms of DNA polymerase have been studied in the basidiomycete Coprinus. DNA polymerase from basidiocarp tissues at zygotene-pachytene stage has been purified 3,500-fold and defined as DNA polymerase b by virtue of its insensitivity to N-ethylmaleimide and by its low molecular weight (76,000). This enzyme has optimal activity at pH 7.0 to 7.5, at 200 mM KCl, and at 25 degrees C incubation temperature. It can use polycytidylic acid-oligo(dG)12-18 as template primer in addition to homodeoxypolymers. The DNA polymerase a is mainly produced in the exponentially growing mycelium. It is sensitive to N-ethylmaleimide and has a temperature optimum at 35 degrees C. At the premeiotic S phase, activities from both polymerase a and polymerase b are found in cell-free extracts. The b enzyme is the only DNA polymerase produced during meiotic prophase. Its assayable activity exhibits two peaks, one at premeiotic S stage and one at pachytene. It is possible that DNA polymerase b is responsible for pachytene repairs involved in recombination.     

Thermostable DNA polymerase from the archaebacterium Sulfolobus acidocaldarius. Purification, characterization and immunological properties

We have purified to near homogeneity a DNA polymerase from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. Sodium dodecyl sulfate gel electrophoresis of the purified enzyme revealed a polypeptide of 100 kDa. On the basis of a Stokes radius of 4.2 nm and a sedimentation coefficient of 6 S, the purified enzyme has an estimated molecular mass of 109 kDa. These results are consistent with the enzyme being a monomer of 100 kDa. In addition a polyclonal antiserum, obtained by injection of the electroeluted 100-kDa polypeptide into a rabbit, specifically neutralized the DNA-polymerase activity. The enzyme is sensitive to both N-ethylmaleimide and 2',3'-dideoxyribosylthymine triphosphate and resistant to aphidicolin. The purified DNA polymerase has neither exonuclease nor primase activities. In our in vitro conditions, the enzyme is thermostable up to 80 degrees C and is active between 55 degrees C and 85 degrees C in the presence of activated calf-thymus DNA.     

The DNA polymerase from the archaebacterium Sulfolobus acidocaldarius: a thermophilic and thermoresistant enzyme which can perform automated polymerase chain reaction

A DNA polymerase purified from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius was used to perform automated DNA amplification at 70 degrees C as well as site directed mutagenesis by Polymerase Chain Reaction (P.C.R.). The yield of amplification performed at optimum MgCl2 concentration for the Taq or the S. acidocaldarius DNA polymerase, for the same DNA target, was equivalent. The ability of S. acidocaldarius DNA polymerase to perform P.C.R. under less stringent requirement of MgCl2 concentration gives this enzyme a non-negligible advantage over the Taq DNA polymerase.     

The DNA polymerase-primase from drosophila melanogaster embryos. Rate and fidelity of polymerization on single-stranded DNA templates

The DNA polymerase activity of the near homogeneous, multisubunit DNA polymerase-primase from Drosophila melanogaster embryos has been compared to Escherichia coli DNA polymerase III core, DNA polymerase III, and DNA polymerase III holoenzyme. The rate of deoxynucleotide incorporation by the Drosophila polymerase on singly primed phi X174 DNA is similar to that observed with equivalent levels of DNA polymerase III holoenzyme in the absence of E. coli single-stranded DNA binding protein. However, analysis of the DNA products indicates that the Drosophila polymerase is less processive than DNA polymerase III holoenzyme, and closely resembles DNA polymerase III. The Drosophila polymerase-primase contains neither 3'-5' exonuclease nor RNase H-like activities, and catalyzes no significant pyrophosphate exchange. There is a low level of DNA-dependent ATPase activity which can be eliminated by a second glycerol gradient sedimentation (Kaguni, L.S., Rossignol, J.-M., Conaway, R.C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 2221-2225). Although lacking a 3'-5' exonuclease, the replication fidelity of the D. melanogaster polymerase is similar to that of E. coli DNA polymerase III holoenzyme which possesses such an activity.     

Isolation and properties of the SuaI restriction endonuclease from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius

A new restriction endonuclease SuaI was isolated from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. The enzyme is an isoschizomer of BspR1; it recognizes tetranucleotide GGCC and cleaves DNA in the center of this sequence. SuaI requires Mg2+, the optimal concentration being 6 mM. KCl at concentrations above 25 mM significantly inhibits the enzyme activity. The pH optimum lies within the range of 6--7 at 70 degrees C, the temperature optimum is at 70--75 degrees C. The enzyme is highly stable at temperatures up to 80 degrees C. DNA of S. acidocaldarius is not cleaved by the enzyme.     

On the association of reverse transcriptase with polynucleotide templates during catalysis.

The association of avian myeloblastosis virus (AMV) DNA polymerase with polynucleotide templates during catalysis has been studied. During the course of polymerization, different template-primer complexes were added and the ability of the enzyme to switch from one polynucleotide template to another was determined. At 37 degrees C as well as at 4 degrees C, the polymerase is able to switch from certain template-primer complexes to others. For example, the addition of poly(A)-oligo(dT) during the course of synthesis with poly(C)-oligo(dG) results in the immediate cessation of dGMP polymerization and the start of dTMP polymerization without any lag. Early during the course of polymerization, the size of the product, as determined by alkaline sucrose gradient centrifugation, is, in part, a function of the ratio of the template-primer complex to the enzyme. These cumulative experiments indicate that catalysis on polynucleotide templates with avian myeloblastosis virus DNA polymerase under the conditions tested is not processive in a classical sense. Similar to cellular DNA polymerases the enzyme can shift from one template-primer to another. Using autoradiography after gel electrophoresis to estimate the product size, it can be calculated that the enzyme switches from one template to another within 0.25 min at 37 degrees C which corresponds to the incorporation of greater than 25 nucleotides. At 4 degrees C, switching can be calculated to occur in less than three nucleotide addition steps. Thus, with certain homopolymers, conditions can be found by which AMV DNA polymerase can switch from one template-primer complex to another, perhaps after each nucleotide addition step.     

A memory effect in DNA replication

A study of the polymerization/excision ratio in the replication of poly(dA), primed with oligo(dT), was carried out with E. coli DNA polymerase I, at various primer and enzyme concentrations. The variations in this ratio suggest that 1) the DNA polymerase is able to switch between two states of low and high exonuclease activities and 2) after dissociating from the template, the DNA polymerase drifts towards the low exonuclease state. The recovery of the high exonuclease state would require several successive incorporations.     

A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates.

In analogy to the Escherichia coli replicative DNA polymerase III we define two forms of DNA polymerase alpha: the core enzyme and the holoenzyme. The core enzyme is not able to elongate efficiently primed single-stranded DNA templates, in contrast to the holoenzyme which functions well on in vivo-like template. Using these criteria, we have identified and partially purified DNA polymerase alpha holoenzyme from calf thymus and have compared it to the corresponding homogeneous DNA polymerase alpha (defined as the core enzyme) from the same tissue. The holoenzyme is able to use single-stranded parvoviral DNA and M13 DNA with a single RNA primer as template. The core enzyme, on the other hand, although active on DNAs treated with deoxyribonuclease to create random gaps, is unable to act on these two long, single-stranded DNAs. E. coli DNA polymerase III holoenzyme also copies the two in vivo-like templates, while the core enzyme is virtually inactive. The homologous single-stranded DNA-binding proteins from calf thymus and from E. coli stimulate the respective holoenzymes and inhibit the core enzymes. These results suggest a cooperation between a DNA polymerase holoenzyme and its homologous single-stranded DNA-binding protein. The prokaryotic and the mammalian holoenzyme behave similarly in several chromatographic systems.     

The effect of template secondary structure on vaccinia DNA polymerase.

Vaccinia virus DNA polymerase will utilize a substrate consisting of phi X174 DNA primed with a strand of a unique restriction fragment, but the reaction is inefficient. Examination of the reaction products by alkaline agarose gel electrophoresis revealed a few discrete fragments, each corresponding to an extended primer strand. This result implies that specific barriers exist on the phi X174 template which impede, but do not completely halt, the progress of the enzyme. Only a few per cent of the template molecules were completely copied. Similar findings were reported by Sherman and Gefter using Escherichia coli DNA polymerase II and fd DNA (J. Mol. Biol. (1976) 103, 61-76). Several observations suggest that the barriers are regions of template secondary structure. Some barriers are more effective than others, and they increase in both effectiveness and number as the temperature is decreased. The same barriers are observed with T4 DNA polymerase, but none are detected with E. coli DNA polymerase I. Finally, the major barriers are located in regions of the phi X174 sequence known to contain hairpin structures of relatively high stability. The exact stopping point of one of the major barriers is within the duplex stem of a hairpin structure. These results show that DNA polymerases are a useful probe of the secondary structure of a single-stranded DNA.     

Spectroscopic analysis of the interaction of Escherichia coli DNA-dependent RNA polymerase with T7 DNA and synthetic polynucleotides.

We have studied the circular dichroism and ultraviolet difference spectra of T7 bacteriophage DNA and various synthetic polynucleotides upon addition of Escherichia coli RNA polymerase. When RNA polymerase binds nonspecifically to T7 DNA, the CD spectrum shows a decrease in the maximum at 272 but no detectable changes in other regions of the spectrum. This CD change can be compared with those associated with known conformational changes in DNA. Nonspecific binding to RNA polymerase leads to an increase in the winding angle, theta, in T7 DNA. The CD and UV difference spectra for poly[d(A-T)] at 4 degrees C show similar effects. At 25 degrees C, binding of RNA polymerase to poly[d(A-T)] leads to hyperchromicity at 263 nm and to significant changes in CD. These effects are consistent with an opening of the double helix, i.e. melting of a short region of the DNA. The hyperchromicity observed at 263 nm for poly[d(A-T)] is used to determine the number of base pairs disrupted in the binding of RNA polymerase holoenzyme. The melting effect involves about 10 base pairs/RNA polymerase molecule. Changes in the CD of poly(dT) and poly(dA) on binding to RNA polymerase suggest an unstacking of the bases with a change in the backbone conformation. This is further confirmed by the UV difference spectra. We also show direct evidence for differences in the template binding site between holo- and core enzyme, presumably induced by the sigma subunit. By titration of the enzyme with poly(dT) the physical site size of RNA polymerase on single-stranded DNA is approximately equal to 30 bases for both holo- and core enzyme. Titration of poly[d(A-T)] with polymerase places the figure at approximately equal to 28 base pairs for double-stranded DNA.     

Further biochemical characterization of wheat DNA primase: possible functional implication of copurification with DNA polymerase A.

DNA primase has been partially purified from wheat germ. This enzyme, like DNA primases characterized from many procaryotic and eucaryotic sources, catalyses the synthesis of primers involved in DNA replication. However, the wheat enzyme differs from animal DNA primase in that it is found partially associated with a DNA polymerase which differs greatly from DNA polymerase alpha. Moreover, the only wheat DNA polymerase able to initiate on a natural or synthetic RNA primer is DNA polymerase A. In this report we describe in greater detail the chromatographic behaviour of wheat DNA primase and its copurification with DNA polymerase A. Some biochemical properties of wheat DNA primase such as pH optimum, Mn + 2 or Mg + 2 optima, and temperature optimum have been determined. The enzyme is strongly inhibited by KCI, cordycepine triphosphate and dATP, and to a lesser extent by cAMP and formycine triphosphate. The primase product reaction is resistant to DNAse digestion and sensitive to RNAse digestion. Primase catalyses primer synthesis on M13 ssDNA as template allowing E.coli DNA polymerase I to replicate the primed M13 single-stranded DNA leading to double-stranded M13 DNA (RF). M13 replication experiments were performed with wheat DNA polymerases A, B, CI and CII purified in our laboratory. Only DNA polymerase A is able to recognize RNA-primed M13 ssDNA.     

Mammalian cyclin/PCNA (DNA polymerase delta auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III.

Human cyclin/PCNA (proliferating cell nuclear antigen) is structurally, functionally, and immunologically homologous to the calf thymus auxiliary protein for DNA polymerase delta. This auxiliary protein has been investigated as a stimulatory factor for the nuclear DNA polymerases from S. cerevisiae. Calf cyclin/PCNA enhances by more than ten-fold the ability of DNA polymerase III to replicate templates with high template/primer ratios, e.g. poly(dA).oligo(dT) (40:1). The degree of stimulation increases with the template/primer ratio. At a high template/primer ratio, i.e. low primer density, cyclin/PCNA greatly increases processive DNA synthesis by DNA polymerase III. At low template/primer ratios (e.g. poly(dA).oligo(dT) (2.5:1), where addition of cyclin/PCNA only minimally increases the processivity of DNA polymerase III, a several-fold stimulation of total DNA synthesis is still observed. This indicates that cyclin/PCNA may also increase productive binding of DNA polymerase III to the template-primer and stabilize the template-primer-polymerase complex. The activity of yeast DNA polymerases I and II is not affected by addition of cyclin/PCNA. These results strengthen the hypothesis that yeast DNA polymerase III is functionally analogous to the mammalian DNA polymerase delta.     

Characterization of revertants derived from a mouse DNA temperature-sensitive mutant strain, tsFT20, which contains heat-labile DNA polymerase alpha activity

One spontaneous and four N-methyl-N'-nitro-N-nitrosoguanidine-induced revertants of a mouse FM3A mutant, tsTF20, which has heat-labile DNA polymerase alpha activity and cannot grow at 39 degrees C, were isolated and characterized with respect to the thermolability of their DNA polymerase alpha activity, the intracellular level of enzyme activity, growth rate, cell cycle progression, and frequency of initiation of DNA replication at the origin of replicons. DNA polymerase alpha activity in the extracts from the revertant cells showed partial recovery of heat stability. The intracellular level of enzyme activity of the revertant cells was lower than that of wild-type cells even at 33 degrees C. The level of enzyme activity in the revertant cells decreased considerably after a temperature upshift to 39 degrees C, but the DNA synthesizing ability of these cells did not decrease as much as the level of enzyme activity. The growth rates of the wild-type and revertant lines were almost the same at 33 degrees C. At 39 degrees C, the rate for the wild-type increased considerably compared to that at 33 degrees C, while little difference in the growth rates of the revertant lines was observed at the two temperatures. Therefore, the doubling times of the revertant cells were relatively increased compared to those of wild-type cells cultured at the restrictive temperature. Flow microfluorometric analysis and cell cycle analysis to measure labeled mitosis revealed that the increase in the doubling time was due mainly to the increase in the duration of the S phase. Analysis of the center-to-center distance between replicons by DNA fiber autoradiography indicated that the frequency of replicon initiation per unit length DNA at a given time was reduced in the revertant cells growing at 39 degrees C.     

Identification of a beta-like DNA polymerase activity in bovine heart mitochondria

A new DNA polymerase activity, distinct from DNA polymerase gamma, has been identified in bovine heart mitochondria. First detected among proteins isolated in a complex with mitochondrial DNA, the DNA polymerase activity has been partially purified 47,000-fold. Enzyme activity separates from DNA polymerase gamma on several chromatographic columns and appears to copurify with a 38 +/- 2-kDa polypeptide. Unlike DNA polymerase gamma, this enzyme is relatively resistant to inhibition by N-ethylmaleimide and dideoxynucleotides, has moderately low monovalent and high divalent cation requirements, and possesses 20-fold-higher apparent K(m) values for deoxynucleotides. The enzyme polymerizes deoxynucleotides onto a primed template DNA in a relatively nonprocessive fashion and lacks a detectable 3' to 5' exonuclease activity. Many of these characteristics resemble a beta-like mitochondrial DNA polymerase previously identified in, and considered unique to, trypanosomes. We propose that the bovine and trypanosomal enzymes are related and represent a new class of ubiquitous mitochondrial DNA polymerases.