The anomalous gel migration of a stable cruciform: temperature and salt dependence, and some comparisons with curved DNA.

We have made an analysis of the gel electrophoretic properties of a pseudo-cruciform fragment, a linear DNA molecule containing a stable cruciform. The migration of this construct was analysed in polyacrylamide gels at a various temperatures in the range 5 degrees to 55 degrees C, and in the presence of NaCl, MgCl2 or ethidium bromide. The magnitude of the anomalous migration (retardation) was almost temperature independent up to 40 degrees C, but decreased strongly beyond this point, extrapolating to normal migration at 70 degrees C. Addition of salts reduced the anomaly. This took the form of a continuous reduction in anomalous migration with the addition of NaCl up to 60 mM, while with MgCl2 there was a sharp reduction in the anomaly to a constant value which is reached by 10 mM. Under these conditions, moreover, the migration of the fragment became almost temperature-independent over the entire range. These results have been interpreted to reflect the influence of ion binding at the four-way junction on the relative disposition of the cruciform arms. The detailed electrophoretic properties of the pseudo-cruciform are in marked contrast to those of sequence-directed curved DNA fragments. In particular, the response to the addition of 1 microgram/ml ethidium bromide offers a convenient method for distinguishing between anomalous retardation arising from curvature (greatly reduced anomaly) or a cruciform junction (enhanced anomaly).     

Suppression of electrophoretic anomaly of bent DNA segments by the structural property that causes rapid migration.

Intrinsic DNA curvature is speculated to be a common feature of all satellite DNA sequences and may aid in the tight winding of DNA in constitutive heterochromatin. Several satellite DNAs, however, show unusually rapid migration in non-denaturing polyacrylamide gels, which is just the opposite behavior of that shown by curved DNA structures. Employing bovine satellite I DNA monomer, we attempted to understand the molecular mechanism of 'rapid migration'. The phenomenon of rapid migration was temperature-dependent and to a small extent polyacrylamide-concentration-dependent. Physiological or near-physiological concentrations of Mg2+and Ca2+ions bent the rapid migrating DNA segment. Predominance of purine-purine base stacking over purine-pyrimidine in nucleotide sequence was strongly indicated to be the cause of the rapid migration. Furthermore, they seemed to be implicated in the formation of induced DNA bend. We also found that the satellite I monomer contains an intrinsic DNA curvature as do many other satellites. Heretofore, the rapid migration property has concealed the presence of curvature.     

A magnesium-induced conformational transition in the loop of a DNA analog of the yeast tRNA(Phe) anticodon is dependent on RNA-like modifications of the bases of the stem.

Two single-stranded DNA heptadecamers corresponding to the yeast tRNA(Phe) anticodon stem-loop were synthesized, and the solution structures of the oligonucleotides, d(CCAGACTGAAGATCTGG) and d(CCAGACTGAAGAU-m5C-UGG), were investigated using spectroscopic methods. The second, or modified, base sequence differs from that of DNA by RNA-like modifications at three positions; dT residues were replaced at positions 13 and 15 with dU, and the dC at position 14 with d(m5C), corresponding to positions where these nucleosides occur in tRNA(Phe). Both oligonucleotides form intramolecular structures at pH 7 in the absence of Mg2+ and undergo monophasic thermal denaturation transitions (Tm = 47 degrees C). However, in the presence of 10 mM Mg2+, the modified DNa adopted a structure that exhibited a biphasic "melting" transition (Tm values of 23 and 52 degrees C) whereas the unmodified DNA structure exhibited a monophasic denaturation (Tm = 52 degrees C). The low-temperature, Mg(2+)-dependent structural transition of the modified DNA was also detected using circular dichroism (CD) spectroscopy. No such transition was exhibited by the unmodified DNA. This transition, unique to the modified DNA, was dependent on divalent cations and occurred most efficiently with Mg2+; however, Ca2+ also stabilized the alternative conformation at low temperature. NMR studies showed that the predominant structure of the modified DNA in sodium phosphate (pH 7) buffer in the absence of Mg2+ was a hairpin containing a 7-nucleotide loop and a stem composed of 3 stable base pairs. In the Mg(2+)-stabilized conformation, the loop became a two-base turn due to the formation of two additional base pairs across the loop.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA curvature in native and modified EcoRI recognition sites and possible influence upon the endonuclease cleavage reaction

The ligation of a decadeoxynucleotide containing the EcoRI recognition site forms a series of multimers which appear to be curved based on observed anomalous gel migration in polyacrylamide gels. The degree of DNA curvature present in the recognition sequence, based upon the observed migration anomaly, can be altered by modifications to the purine functional groups at the 2- and 6-positions. Deletion of the guanine 2-amino group, occurring in the minor groove of the B-DNA helix, is most effective in increasing the observed DNA curvature. Conversely, the displacement of an amino group from the major groove to the minor groove eliminates curvature. DNA curvature is also modulated by the exocyclic group at the purine 6-position with decreasing curvature observed when changing the amino group to a carbonyl or proton substituent. Differences in the kinetic parameters characterizing the cleavage reaction by the endonuclease for many of the modified sequences are the result of modifications of functional groups in the major groove, which are likely to contact the endonuclease during catalysis. However, with two examples, significant decreases in the observed specificity constant (kcat/Km), characterizing the protein-nucleic acid interaction, cannot be easily explained in terms of such functional group contacts. It is more likely in these cases that the functional group modifications affect the efficiency of the endonuclease-DNA interaction by modulation of the structure of the double-stranded DNA helix. With both examples, modifications have been made to minor groove substituents. The extent of DNA curvature is increased significantly for one and decreased for the other, compared with that observed for the native recognition site. The results suggest that curvature of the DNA helix axis is an intrinsic property of the d(GAATTC) sequence which helps to optimize the protein-nucleic acid interactions observed for the EcoRI restriction endonuclease.     

Branched nanowire based guanine rich oligonucleotides.

Self-assembly and aggregation of guanine rich sequences can provide useful insights into DNA nanotechnology and telomeric structure and function. In this paper, we designed a guanine rich sequence d(GGCGTTTTGCGG). We found that it can form stable structure in appropriate condition and it exhibits an anomalous CD spectra. This structures can be imaged in ambient environment with a Nanoscope III AFM (Digital Instruments). We found it forms branch structure and long multistrand DNA nanowire after incubation at 37 degrees C for 6-12 hours in 25 mM TE (pH=8.0) + 5 mM Mg2+ + 50 mM K+. The ability to self-assemble into branches and long wires not only clearly demonstrate its potential as scaffold structures for nanotechnology, but also give aids to understand telomeric structure further. We have proposed a model to explain how these structures formed.     

Sequence periodicity in nucleosomal DNA and intrinsic curvature

Background

Most eukaryotic DNA contained in the nucleus is packaged by wrapping DNA around histone octamers. Histones are ubiquitous and bind most regions of chromosomal DNA. In order to achieve smooth wrapping of the DNA around the histone octamer, the DNA duplex should be able to deform and should possess intrinsic curvature. The deformability of DNA is a result of the non-parallelness of base pair stacks. The stacking interaction between base pairs is sequence dependent. The higher the stacking energy the more rigid the DNA helix, thus it is natural to expect that sequences that are involved in wrapping around the histone octamer should be unstacked and possess intrinsic curvature. Intrinsic curvature has been shown to be dictated by the periodic recurrence of certain dinucleotides. Several genome-wide studies directed towards mapping of nucleosome positions have revealed periodicity associated with certain stretches of sequences. In the current study, these sequences have been analyzed with a view to understand their sequence-dependent structures.

Results

Higher order DNA structures and the distribution of molecular bend loci associated with 146 base nucleosome core DNA sequence from C. elegans and chicken have been analyzed using the theoretical model for DNA curvature. The curvature dispersion calculated by cyclically permuting the sequences revealed that the molecular bend loci were delocalized throughout the nucleosome core region and had varying degrees of intrinsic curvature.

Conclusions

The higher order structures associated with nucleosomes of C.elegans and chicken calculated from the sequences revealed heterogeneity with respect to the deviation of the DNA axis. The results points to the possibility of context dependent curvature of varying degrees to be associated with nucleosomal DNA.

     

The specific binding, bending, and unwinding of DNA by RsrI endonuclease, an isoschizomer of EcoRI endonuclease

To determine whether RsrI endonuclease recognizes and cleaves the sequence GAATTC in duplex DNA similarly to its isoschizomer EcoRI we initiated a functional comparison of the two enzymes. Equilibrium binding experiments showed that at 20 degrees C RsrI endonuclease binds to specific and nonspecific sequences in DNA with affinities similar to those of EcoRI. At 0 degrees C the affinity of RsrI for its specific recognition sequence is reduced 7-fold whereas the affinity for noncanonical sequences remains relatively unchanged. Unlike EcoRI, incubation of RsrI endonuclease with N-ethylmaleimide inactivates the enzyme; however, preincubation with DNA prevents the inactivation. The N-ethylmaleimide-treated enzyme fails to bind DNA as assayed by gel mobility shift assays. Comparison of the deduced amino acid sequences of RsrI and EcoRI endonucleases suggests that modification of Cys245 is responsible for the inactivation. Fe(II). EDTA and methidiumpropyl-EDTA.Fe(II) footprinting results indicate that RsrI, like EcoRI, protects 12 base pairs from cleavage when bound to its specific recognition sequence in the absence of Mg2+. RsrI bends DNA by approximately 50 degrees, as determined by measuring the relative electrophoretic mobilities of specific RsrI-DNA complexes with the binding site in the center or near the end of the DNA fragment. This value is similar to that reported for EcoRI. RsrI also unwinds the DNA helix by 25 degrees +/- 5 degrees, a value close to that reported for EcoRI endonuclease. Collectively, these results indicate that the overall structural changes induced in the DNA by the binding of RsrI and EcoRI endonucleases to DNA in the absence of Mg2+ are similar. In the accompanying paper (Aiken, C. R., McLaughlin, L. W., and Gumport, R. I. (1991) J. Biol. Chem. 266, 19070-19078) we present results of studies of RsrI endonuclease using oligonucleotide substrates containing base analogues which suggest differences in the ways the two enzymes cleave DNA.     

Capillary electrophoresis as a technique to analyze sequence-induced anomalously migrating DNA fragments.

Sequence-induced anomalous migration of double-stranded (ds) DNA in native gel electrophoresis is a well known phenomenon. The retardation of migration is more obvious in polyacrylamide compared with agarose gels, and is greatly affected by the concentration of the gel and the temperature. This anomalous migration results in a difference between calculated and actual sizes of the affected DNA fragments. A low viscosity polymer solution (DNA Fragment Analysis Reagent) under investigation for use in dsDNA analysis by capillary electrophoresis is shown to be useful for the visualization of anomalies in migration of dsDNA fragments. Comparable with traditional slab gel systems, the retardation effect, indicative of bent or curved DNA, is strongly dependent on polymer concentration and separation temperature. These dependencies have implications on the accurate sizing of dsDNA fragments with unknown sequences and secondary structures.     

Deoxyuridine triphosphate nucleotidohydrolase induced by herpes simplex virus type 1. Purification and characterization of induced enzyme

A deoxyuridine triphosphate nucleotidohydrolase (dUTPase) which is induced in KB cells infected with herpes simplex virus type 1 (HSV-1) was purified approximately 175-fold using affinity, hydrophobic, adsorption, and ion-exchange chromatography techniques. Of the nucleoside triphosphates commonly found in DNA and RNA, only dUTP acted as a substrate for the enzyme, and the apparent Km was 4 microM. While the HSV-1-induced dUTPase exhibited activity in the absence of added divalent cations, the activity was stimulated by Mg2+ and inhibited by EDTA. The inhibition caused by EDTA was reversed by Mg2+, Co2+, or Mn2+. The HSV-1-induced dUTPase was also inhibited by hydroxymercuribenzoate and to a lesser degree by pyrophosphate but not by orthophosphate. The molecular weight of the enzyme was estimated to be 53,000, and its isoelectric point was 5.8. The enzyme exhibited maximal activity over the pH range of 6.5-8.5. The enzyme was thermolabile at 45 degrees C, exhibiting a t1/2 of 35 min. The HSV-1-induced dUTPase was distinguishable from the KB dUTPase by its elution pattern on the various chromatography matrixes, isoelectric point, migration in polyacrylamide gels, thermostability, and response to divalent cations.     

DNA methylation can enhance or induce DNA curvature.

Oligomers of different palindromic sequences (EcoRI, BamHI, and ClaI linkers) were ligated to form distributions of multimers. These long ligation ladders were methylated using corresponding methylases. The migration of the unmethylated as well as the methylated multimer distributions was analysed in 10% polyacrylamide gels. The migration anomaly of these sequences is interpreted in terms of the curvature of the DNA helix axis. The double-stranded oligomer dCGGAATTCCG is considerably curved in its unmodified form. Its curvature is strongly enhanced when the central dAs are methylated. This result is predicted by a model for DNA curvature. Multimers of the closely related sequence dCGGGATCCCG are straight. When methylated at the central dAs or at the most central dCs, a small curvature of the helix axis is induced. The double-stranded oligomer dCCATCGATGG is straight in its unmodified form as well as when it is methylated. Thus, DNA curvature can be induced or enhanced by methylation. However, DNA methylation at palindromic sequences seems not always to influence the linear path of the DNA helix axis.     

Stability of G,A triple helices.

In this work we selected double-stranded DNA sequences capable of forming stable triplexes at 20 or 50 degrees C with corresponding 13mer purine oligonucleotides. This selection was obtained by a double aptamer approach where both the starting sequences of the oligonucleotides and the target DNA duplex were random. The results of selection were confirmed by a cold exchange method and the influence of the position of a 'mismatch' on the stability of the triplex was documented in several cases. The selected sequences obey two rules: (i) they have a high G content; (ii) for a given G content the stability of the resulting triplex is higher if the G residues lie in stretches. The computer simulation of the Mg2+, Na+and Cl-environment around three triplexes by a density scaled Monte Carlo method provides an interpretation of the experimental observations. The Mg2+cations are statistically close to the G N7 and relatively far from the A N7. The presence of an A repels the Mg2+from adjacent G residues. Therefore, the triplexes are stabilized when the Mg2+can form a continuous spine on G N7.     

Thermal and Mg2+ dependent behavior of pseudouridines at 39th and 55th of yeast tRNAPhe

Pseudouridine psi 55 alone and both psi 55 and psi 39 in yeast tRNAPhe are selectively modified with fluorescent reagent of 4-bromomethyl-7-methoxycoumarin (BMC). The change of fluorescence intensity was measured as a function of temperature and Mg2+ concentration. Fluorescent quenching shows the stacked and unstacked forms of Y base, dependent on Mg2+ concentration. In contrast, Mg2+ had no effect on psi 55-BMC in T psi C loop at 20 degrees C. Fluorescence on titrating Mg2+ exhibited a kind of Mg2+-induced structural collapse at the corner of L-structure. The melting of psi 55-BMC takes place at 70 degrees C in 10mM Mg2+. At very low Mg2+ concentration, melting takes place at 35 degrees C. The melting of psi 39-BMC, located near the anticodon loop, was observed before the unfolding of the whole structure of tRNAPhe. A conformational transition of the anticodon loop takes place at a lower temperature and it is also expected in the quenching experiment of Y base.     

Isolation and properties of a thermostable restriction endonuclease (ENDO R-Bst1503).

A restriction endonuclease was isolated from Bacillus stearothermophilus1503-4R (Bst1503) and purified to homogeneity. The enzyme required Mg2+ ion as a cofactor. Bst1503 exhibited maximal activity between pH 7.5 and 8.0, between 60 and 65 degrees C, and with about 0.2 mM Mg2+. Bst1503 was not inactivated after exposure at 55 or 65 degrees C for up to 10 h. After 2 h of incubation at 70 degrees C, Bst1503 was inactivated by 65%. Bst1503 was rapidly inactivated at 75 degrees C. A single protein-staining band having a molecular weight of 46,000 was observed when Bst1503 was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme was found to exist in two active forms, the predominating form with an S value of 8.3 (180,000) and the second form with an S value of 5.4 (96,000). No conversion between the 8.3S and 5.4S forms was observed after storage. Bst1503 recognized six sites in TP-1C deoxyribonucleic acid (DNA), one site in pSC101 and simian virus 40 DNAs, and three sites in lambdavir DNA. Bst1503 and BamHI were determined to be isoschizomers. The effect of temperatures on the activity and stability of BamHI was determined.     

Temperature and salt dependence of the gel migration anomaly of curved DNA fragments.

A series of oligonucleotides of different sequences have been cloned to study DNA curvature. Several DNA fragments containing these oligonucleotides in various numbers of repeats were analyzed in 10% polyacrylamide gels. A strong gel migration anomaly was found for dA4 sequences; a comparably very small but clearly detectable anomaly was observed for dA3 (both in a repeat length of 10 base-pairs). The temperature and salt (NaCl, MgCl2) dependence of the gel migration anomaly of these DNA fragments was measured. While a similar behaviour of all sequences is observed for the addition of NaCl, the temperature and MgCl2 dependence of the anomaly varies with the oligonucleotide sequence. These data are interpreted in terms of local DNA structure changes induced by changes in the temperature and the MgCl2 concentration which affect the planarity of the curved DNA fragments.     

Nitrosamine-induced cancer: O4-alkylthymine produces sites of DNA hyperflexibility.

The carcinogenic properties of N-nitroso compounds are associated with their ability to alkylate DNA, in particular to form O6-alkylguanine and O4-alkylthymine. DNA duplexes containing either O6-alkylguanine or O4-alkylthymine were synthesized, and each duplex was ligated to form a set of DNAs of increasing length with the alkylated base out of phase (16 base-pairs apart) or in phase (21 base-pairs apart) with the helical repeat of the DNA. The DNA contained the sequence 5' CAA 3', which is the 61st codon of the K-ras gene, because this codon is a preferred site of mutation for a number of carcinogens including the methylating carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1- butanone (NNK). O4-Methylthymine or O4-ethylthymine replaced thymine in either of the two A.T base-pairs of this codon (normally CAA), and O6-methylguanine replaced the guanine in the G.C pair. All the sequences containing O4-alkylthymine exhibited anomalous, slow, gel migration and ligated to form circles of unusually small diameter. In general, the effect was seen when the alkylated base-pair was out of phase with the helical repeat as well as when it was in phase, suggesting that the alkylated base-pair confers flexibility which is largely isotropic, i.e., has no preferred direction, rather than anisotropic flexibility or bending. However, at pH 8.3 the 21-base-pair set containing O4-alkylT.A had significantly greater anomalous migration than the 16-base-pair set, suggesting that the flexibility produced by this base-pair has a significant anisotropic component and thus resembles true bending.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transformation in Escherichia coli: stages in the process.

Transformation experiments with Escherichia coli recipient cells and linear chromosomal deoxyribonucleic acid (DNA) are reported. E. coli can be rendered competent for DNA uptake by a temperature shock (0 degrees C leads to 42 degrees C leads to 0 degrees C) of the recipient cells in the presence of a high concentration of either Ca2+ or Mg2+ ions. Uptake of DNA into a deoxyribonuclease-resistant form, for which the presence of Ca2+ is essential, was possible during the temperature shock but appeared to occur most readily after the heat shock during incubation at 0 degrees C. When DNA was added to cells that had been heat shocked in the presence of divalent cations only, DNA uptake also occurred. This suggests that competence induction and uptake may be regarded as separate stages. Under conditions used to induce competence, we observed an extensive release of periplasmic enzymes, probably reflecting membrane damage induced during development of competence. After the conversion of donor DNA into a deoxyribonuclease-resistant form, transformants could be selected. It appeared that incubation, before plating, of the transformation mixture in a medium containing high Ca2+ and Mg2+ concentrations and supplemented with all growth requirements increased the transformation frequency. This incubation probably causes recovery of physiologically labile cells.     

The role of 5-methylcytidine in the anticodon arm of yeast tRNA(Phe): site-specific Mg2+ binding and coupled conformational transition in DNA analogs.

The tDNA(Phe)AC, d(CCAGACTGAAGAU13m5C14U15GG), with a DNA sequence similar to that of the anticodon stem and loop of yeast tRNA(Phe), forms a stem and loop structure and has an Mg(2+)-induced structural transition that was not exhibited by an unmodified tDNA(Phe)AC d(T13C14T15) [Guenther, R. H., Hardin, C. C., Sierzputowska-Gracz, H., Dao, V., & Agris, P. F. (1992) Biochemistry (preceding paper in this issue)]. Three tDNA(Phe)AC molecules having m5C14, tDNA(Phe)AC d(U13m5C14U15), d(U13m5C14T15), and d(T13,5C14U15), also exhibited Mg(2+)-induced structural transitions and biphasic thermal transitions (Tm approximately 23.5 and 52 degrees C), as monitored by CD and UV spectroscopy. Three other tDNA(Phe)AC, d(T13C14T15), d(U13C14U15), and d(A7;U13m5C14U15) in which T7 was replaced with an A, thereby negating the T7.A10 base pair across the anticodon loop, had no Mg(2+)-induced structural transitions and only monophasic thermal transitions (Tm of approximately 52 degrees C). The tDNA(Phe)AC d(U13m5C14U15) had a single, strong Mg2+ binding site with a Kd of 1.09 x 10(-6) M and a delta G of -7.75 kcal/mol associated with the Mg(2+)-induced structural transition. In thermal denaturation of tDNA(Phe)AC d(U13m5C14U15), the 1H NMR signal assigned to the imino proton of the A5.dU13 base pair at the bottom of the anticodon stem could no longer be detected at a temperature corresponding to that of the loss of the Mg(2+)-induced conformation from the CD spectrum. Therefore, we place the magnesium in the upper part of the tDNA hairpin loop near the A5.dU13 base pair, a location similar to that in the X-ray crystal structure of native, yeast tRNA(Phe).(ABSTRACT TRUNCATED AT 250 WORDS)     

Blood oxygen transport in common map turtles during simulated hibernation

We assessed the effects of cold and submergence on blood oxygen transport in common map turtles (Graptemys geographica). Winter animals were acclimated for 6-7 wk to one of three conditions at 3 degrees C: air breathing (AB-3 degrees C), normoxic submergence (NS-3 degrees C), and hypoxic (PO2=49 Torr) submergence (HS-3 degrees C). NS-3 degrees C turtles exhibited a respiratory alkalosis (pH 8.07; PCO2=7.9 Torr; [lactate]=2.2 mM) relative to AB-3 degrees C animals (pH 7.89; PCO2=13.4 Torr; [lactate]=1.1 mM). HS-3 degrees C animals experienced a profound metabolic acidosis (pH 7.30; PCO2=7.9 Torr; [lactate]=81 mM). NS-3 degrees C turtles exhibited an increased blood O2 capacity; however, isoelectric focusing revealed no seasonal changes in the isohemoglobin (isoHb) profile. Blood O2 affinity was significantly increased by cold acclimation; half-saturation pressures (P50's) for air-breathing turtles at 3 degrees and 22 degrees C were 6.5 and 18.8 Torr, respectively. P50's for winter animals submerged in normoxic and hypoxic water were 5.2 and 6.5 Torr, respectively. CO2 Bohr slopes (Delta logP50/Delta pH) were -0.15, -0.16, and -0.07 for AB-3 degrees C, NS-3 degrees C, and HS-3 degrees C turtles, respectively; the corresponding value for AB-22 degrees C was -0.37. The O2 equilibrium curve (O2EC) shape was similar for AB-3 degrees C and NS-3 degrees C turtles; Hill plot n coefficients ranged from 1.8 to 2.0. The O2EC shape for HS-3 degrees C turtles was anomalous, exhibiting high O2 affinity below P50 and a right-shifted segment above half-saturation. We suggest that increases in Hb-O2 affinity and O2 capacity enhance extrapulmonary O2 uptake by turtles overwintering in normoxic water. The anomalous O2EC shape and reduced CO2 Bohr effect of HS-3 degrees C turtles may also promote some aerobic metabolism in hypoxic water.     

Comparative analysis of pyruvate kinases from the hyperthermophilic archaea Archaeoglobus fulgidus,Aeropyrum pernix,and Pyrobaculum aerophilum and the hyperthermophilic bacterium Thermotoga maritima: unusual regulatory properties in hyperthermophilic archaea

Pyruvate kinases (PK, EC 2.7.1.40) from three hyperthermophilic archaea (Archaeoglobus fulgidus strain 7324, Aeropyrum pernix, and Pyrobaculum aerophilum) and from the hyperthermophilic bacterium Thermotoga maritima were compared with respect to their thermophilic, kinetic, and regulatory properties. PKs from the archaea are 200-kDa homotetramers composed of 50-kDa subunits. The enzymes required divalent cations, Mg2+ and Mn2+ being most effective, but were independent of K+. Temperature optima for activity were 85 degrees C (A. fulgidus) and above 98 degrees C (A. pernix and P. aerophilum). The PKs were highly thermostable up to 110 degrees C (A. pernix) and showed melting temperatures for thermal unfolding at 93 degrees C (A. fulgidus) or above 98 degrees C (A. pernix and P. aerophilum). All archaeal PKs exhibited sigmoidal saturation kinetics with phosphoenolpyruvate (PEP) and ADP indicating positive homotropic cooperative response with both substrates. Classic heterotropic allosteric regulators of PKs from eukarya and bacteria, e.g. fructose 1,6-bisphosphate or AMP, did not affect PK activity of hyperthermophilic archaea, suggesting the absence of heterotropic allosteric regulation. PK from the bacterium T. maritima is also a homotetramer of 50-kDa subunits. The enzyme was independent of K+ ions, had a temperature optimum of 80 degrees C, was highly thermostable up to 90 degrees C, and had a melting temperature above 98 degrees C. The enzyme showed cooperative response to PEP and ADP. In contrast to its archaeal counterparts, the T. maritima enzyme exhibited the classic allosteric response to the activator AMP and to the inhibitor ATP. Sequences of hyperthermophilic PKs showed significant similarity to characterized PKs from bacteria and eukarya. Phylogenetic analysis of PK sequences of all three domains indicates a distinct archaeal cluster that includes the PK from the hyperthermophilic bacterium T. maritima.