From the
Centre for Crystallographic Studies, Department of Chemistry, University of Copenhagen, Universitetsparken 5, DK-2100 Copenhagen Ø, Denmark, the ¶Department of Biological Chemistry, Institute of Molecular Biology, University of Copenhagen, Sølvgade 83 H, DK-1307 Copenhagen K, Denmark, and the
Department of Biochemistry, Lund University, P.O. Box 124, S-221 00 Lund, Sweden
Received for publication, December 20, 2002 , and in revised form, March 28, 2003.
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ABSTRACT |
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INTRODUCTION |
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The enzyme dCTP deaminase was first characterized from the enteric bacterium, Salmonella typhimurium (4). The oligomeric enzyme showed sigmoid saturation kinetics, and the sigmoidity increased in the presence of dTTP, the end product of the pathway. Product inhibition by dUTP was also significant. Recent analyses of the overproduced E. coli enzyme showed that the enzyme does not contain bound metal,1 in contrast to most other enzymes capable of deaminating the cytosine ring like (deoxy)cytidine deaminase, dCMP deaminase, and RNA editing (C to U) enzymes, which contain firmly bound catalytic zinc, and cytosine deaminase, which contains iron.
dUTPase is a ubiquitous and well characterized enzyme with the essential function of preventing uracil misincorporation into DNA. Amino acid sequence analyses display five conserved motifs within the relatively short subunits of about 150 residues (although exceptions occur among viruses and parasites). Four quite similar crystal structures of dUTPases, from eubacteria, retroviruses, and man, show a unique homotrimeric arrangement where each of the three active sites are formed by contributions from all three subunits: motifs 1, 2, and 4 from subunit A, motif 3 from subunit B, and motif 5 from subunit C (5, 6, 7, 8). Despite the highly intertwined trimeric structures, kinetic studies did not indicate any sign of cooperativity (9, 10).
The two enzymes, dCTP deaminase and dUTPase, catalyze two consecutive steps in deoxynucleotide metabolism (dCTP>dUTP>dUMP), and an evolutionary relationship, also comprising pseudouridine synthases, was first suggested by Koonin (11). Homology was especially noted for the sequence referred to as motif 3 in the dUTPases, where the residues form a -hairpin structure responsible for binding the deoxyuridine moiety. All of the enzymes bind and transform pyrimidine nucleotides, and the relationship is supported by similarities in amino acid sequence to an extent that has confused the annotations of genes from sequenced archaeal genomes. Most archaeons had, until recently, two genes annotated as dCTP deaminases but no dUTPase.
Two archaeal dUTPases have, so far, been conclusively identified. The first was reported for the Sulfolobus islandicus rod-shaped virus SIRV (12), and the second was from Pyrococcus furiosis (13). By analysis of the first sequenced archaeal genome, that of Methanocaldococcus jannaschii, Prangishvili et al. (12) suggested that the genes MJ1102 and MJ0430 encoded a dUTPase and a dCTP deaminase, respectively. Because archaeal dCTP deaminase and dUTPase sequences appeared to differ from counterparts in the two other domains of life, eubacteria and eukaryotes, we decided to determine the catalytic capacity of the MJ1102 and MJ0430 gene products. In the present work, we confirm the previous assignment (12) and show that the MJ1102 gene product is a specific dUTPase, whereas the MJ0430 protein is a bifunctional enzyme possessing both dCTP deaminase and dUTPase activity.
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EXPERIMENTAL PROCEDURES |
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Strains, Plasmids, and Gene Cloning
E. coli XL1Blue (Promega) was used for all clonings, and E. coli BL21(DE3) was used for recombinant protein overproduction. The two genes MJ1102 and MJ0430 were amplified by PCR from genomic M. jannaschi DNA obtained from the American Type Culture Collection (ATCC 43067) with primers introducing restriction endonuclease sites for NdeI and BamHI before the start codon and after the stop codon, respectively. After purification on QIAquick mini columns (Qiagen) and digestion with NdeI and BamHI, the fragments (514 and 638 bp) were cloned into plasmid pET-3a (15), yielding pET-3a/MJ1102 and pET-3a/MJ0430. DNA sequencing of both strands of the cloned inserts revealed no differences when compared with the published sequences (16). The two constructs were transformed into E. coli BL21(DE3) and BL21(DE3)/pRI952. Plasmid pRI952 (17) carries genes for the rare tRNAIle (AUA) and tRNAArg (AGA and AGG) codons and for resistance to chloramphenicol (CmR).
Small Scale Expression
5-ml cultures from freshly transformed cells were grown at 37 °Cin Luria Bertani medium (18) containing the appropriate antibiotics (100 µg of ampicillin/ml for strains harboring pET-3a plasmids and 30 µg of chloramphenicol/ml for strains harboring pRI952). At an optical density at 600 nm of 1.0, isopropyl-
-D-thiogalactopyranoside was added to the medium at a final concentration of 0.5 mM, and incubation was continued with shaking at 37 °C overnight. The cells were harvested by centrifugation, suspended in 50 mM potassium phosphate, pH 6.8, and frozen and thawed three times, yielding permeabilized cells.
Purification of Protein
The chromatography steps were carried out at room temperature, and the centrifugations (4 °C) were performed in a Sorvall SS-34 rotor.
dCTP DeaminaseCells from three induced 250-ml cultures of the strain carrying pET-3a/MJ0430, grown as described above, were suspended in 150 ml of extraction buffer (20 mM Tris-HCl, pH 9.0, 5 mM dithiothreitol, 1 mM EDTA, 1 mM ethylene glycol bis(-aminoethyl ether) N,N'-tetraacetic acid, and 1 mM phenylmethylsulfonyl fluoride). The cell suspension was incubated at 75 °C for 30 min and centrifuged at 11 000 rpm for 30 min. The cleared extract was loaded on a Q-Sepharose column (2.6 x 12 cm) previously equilibrated with buffer A (20 mM Tris-HCl, pH 9.0, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride). After washing the column with 100 ml of buffer A, elution was performed using a linear NaCl gradient from 0 to 0.25 M in 500 ml of buffer A. Peak fractions, essentially free of contaminating protein as judged by SDS-PAGE, were loaded and chromatographed on a Red-Sepharose column (1.0 x 9 cm), equilibrated in buffer B (25 mM potassium phosphate, pH 7.5, 0.5 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride). UV-absorbing material was observed in the flow-through and during wash of the column (by 15 ml of buffer B), and elution was achieved by a gradient from 0 to 2 M KCl in 65 ml of buffer B. Peak fractions (eluted at 0.81.0 M KCl) were concentrated to about 20 mg/ml by ultrafiltration in a Centriprep tube (Amicon, Inc.) and stored a 4 °C. The enzyme was quantified by using its theoretical extinction coefficient (A280320) 25,010 M1 cm1, and the final yield from 750 ml of cell culture was estimated to be 45 mg.
dUTPaseCells from an induced 25-ml culture of strain BL21(DE3) carrying pET-3a/MJ1102 were suspended in 5 ml of extraction buffer, heat-treated, and centrifuged as above. The supernatant was applied on a DE-52 column (2 ml), and the protein was eluted in isocratic steps by NaCl in buffer A. The purest fraction, eluted with 100 mM NaCl, was desalted on a PD-10 column equilibrated with 0.1 M KCl, 5 mM MgCl2, and 250 µM Bicine,2 pH 8.2. The protein concentration was assessed by the method of Kalb and Bernlohr (19) from absorbance at 230 and 260 nm.
Gel Electrophoresis, Protein Blotting, and Mass Spectrometry
SDS-PAGE containing 15% polyacrylamide in the resolving gel was run using a Mini Protean II equipment from Bio-Rad. Samples of overproduced protein from permeabilized cells were blotted onto a poly-vinylidene difluoride membrane (Polyscreen; PerkinElmer Life Sciences) according to standard procedures (20). Edman degradation was performed by Ingrid Dahlqvist (Clinical Chemistry Laboratory at Malmö University Hospital) on an Applied Biosystems 494 gas phase sequenator. Samples for mass spectrometry were transferred to 0.1% trifluoroacetic acid, and the analyses were performed by Michael Nielsen and Krestine Greve at the Department of Protein Chemistry of Copenhagen University using a matrix-assisted laser desorption/ionization time-of-flight mass spectrometry instrument (Bruker Biflex III). The protein samples were mixed with an equal volume of matrix solution (sinapinic acid in 35% acetonitrile and 0.1% trifluoroacetic acid).
Enzyme Assays
All assays were conducted at 25 °C unless otherwise stated, and the activity parameter for purified enzyme is given as turnover number (s1). For the dUTPase assay on permeabilized cells, activity is expressed as mmol of dUTP hydrolyzed per min per liter of bacterial culture.
dCTP Deaminase Assay 1The reaction volume was 50 µl in 50 mM potassium phosphate, pH 6.8, and 5 mM MgCl2 (assay buffer). Enzyme concentration was typically 2 µM, and the reaction was started by the addition of 1 mM [3H]dCTP (2 µCi/µmol). At intervals, 5-µl samples of the reaction mixture were withdrawn and applied to TLC plates. Prior to application, 20 nmol each of dUTP, dCTP, dUMP, and dCMP and 200 nmol of EDTA were added to the start points 3 cm above the lower edge of the plate. The plates were developed stepwise in one dimension as follows: (i) 1 M HAc for 1 min, (ii) 0.9 M HAc and 0.3 M LiCl to 3 cm above the start line, and (iii) 1 M HAc and 1.3 M LiCl to 14 cm above the start line. After development, the plates were washed in absolute methanol for 5 min and dried with hot air, and the marker spots were identified by UV light. The spots were cut out and placed in scintillation vials, and 0.7 ml of 2 M NH3 was added to elute nucleotides from the layers before determination of their radioactivity by liquid scintillation counting. The Rf values for dUTP, dCTP, dUMP, and dCMP were 0.25, 0.34, 0.64, and 0.78, respectively. dUDP and dCDP, which may occur as contaminants in the triphosphate markers, showed Rf values of 0.47 and 0.54, respectively.
dCTP Deaminase Assay 2This assay, mainly used to locate active fractions during purification, is based on the large shift in absorbance at 290 nm upon deamination of the cytosine ring to uracil at acidic pH (E290 = 10,300 M1 cm1, pH 2.0). The reaction mixtures contained 2 mM dCTP in assay buffer, and the reaction was started by the addition of enzyme. After 30 and 60 min, 50 µl of the reaction mixture was added to 1.0 ml of 0.5 M perchloric acid, and the absorbance at 290 nm was measured.
dCTP Deaminase Assay 3This assay was used for continuous measurements. In kinetic experiments the dCTP concentration was varied between 10 and 500 µM. Absorbances were recorded at 282 or 290 nm in assay buffer (pH 6.8) in which the E282 and
E290 for the deamination of dCTP were 3500 and 1900 M1 cm1, respectively.
dUTPase Assay 1Permeabilized cells were assayed for dUTPase activity with [3H]dUTP as previously described (21, 22). The assay mixture contained 0.9 mM dUTP in 0.2 M potassium phosphate, pH 6.5, 10 mM MgCl2, and 10 mM dithiothreitol, and the assay temperature was 80 °C. After 10 min, the reaction product [3H]dUMP was separated from the substrate by TLC in 1.0 M formic acid containing 0.5 M LiCl, and the number of counts in the dUMP and dUTP spots was determined (22).
dUTPase Assay 2To determine the initial rate and Km for dUTP, the spectrophotometric method of Larsson et al. (9) was used. Protons released by hydrolysis of dUTP to dUMP were monitored in a weakly buffered solution in the presence of a pH indicator. Prior to kinetic measurements, the enzyme was passed through a PD-10 column (Pharmacia Corp.) equilibrated with 0.1 M KCl, 5 mM MgCl2, and 250 µM Bicine, pH 8.2. The reactions were performed in 0.1 M KCl, 5 mM MgCl2, and 250 µM Bicine, pH 8.2, with 25 µM cresol red as indicator, and the decrease in absorbance at 573 nm was recorded. During the course of the reaction, the decrease was not more than 0.2 absorbance units, corresponding to a decrease of less than 0.2 pH units within the pH range of 7.88.2. The reaction trace, which represents a large number of initial rate determinations, was evaluated using the integrated Michaelis-Menten equation and nonlinear regression (9). In this study, a Windows-based program, IMMFitter,3 which also employs the Lambert omega function, was used (23).
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RESULTS |
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Isopropyl--D-thiogalactopyranoside-induced overnight cultures, carrying the constructs pET-3a/MJ0430 and pET-3a/MJ1102 in both E. coli BL21(DE3) and in its pRI952 derivative, were analyzed by SDS-PAGE. As a negative control, pET-3a without insert was used. As shown in Fig. 1, the MJ1102 gene caused induction of an 18-kDa protein (lane 5) in agreement with the deduced size of the open reading frame carried on the recombinant plasmid. The presence of the MJ0430 gene induced a 28-kDa peptide (lane 4), which was significantly larger than the 23.4 kDa predicted from the nucleotide sequence.
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In the BL21(DE3)/pRI952 host, the induced bands at 18 and 28 kDa were likewise observed, although at somewhat lower intensities (Fig. 1, lanes 1 and 2). In addition, a strong band at 25 kDa was reproducibly observed in conjunction with the pET-3a/MJ1102 construct (Fig. 1, lane 2). Most likely, this band corresponded to the chloramphenicol acetyltransferase subunit (25 kDa) encoded by the cat gene present on pRI952. However, we have no explanation for why it was not observed with the MJ0430 construct or the negative control (Fig. 1, lanes 1 and 3).
Edman DegradationThe three strongly overproduced polypeptides seen in Fig. 1 were subjected to Edman degradation. From the N-terminal sequence, MEKKITGYT, the 25-kDa protein (lane 2) was identified as chloramphenicol acetyltransferase. The sequence MILSDKDIID was obtained from the 28-kDa protein (lane 4) in agreement with the sequence encoded by the MJ0430 gene. The N terminus (MVVKLMIIGA) of the 18-kDa protein (lane 5) conformed to the MJ1102 gene, but about one-third of the protein molecules retained the methionine (143 pmol) as the first residue, whereas the rest started with valine (237 pmol), generating two overlaying but interpretable sequences through the degradation. According to the general rules for processing (24), removal of an N-terminal methionine is to be expected when followed by a valine residue. The inhomogeneiety observed may cause problems for a future determination of the three-dimensional structure.
Assays of Permeabilized CellsPermeabilized cells were assayed for dUTPase activity using Assay 1. The incubations were carried out at 80 °C, a temperature at which the endogeneous E. coli dUTPase is inactive. Unexpectedly, both pET-3a/MJ0430 and pET-3a/MJ1102 supported dUTPase activity (Table I). The presence of plasmid pRI952 did not improve the level of activity for the two constructs, and it was abandoned in all further studies.
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The ability of the various constructs to mediate deamination of dCTP was tested by incubating the permeabilized cells with dCTP at 80 °C for 1 h. A complete shift, from a cytosine to a uracil spectrum, was observed with extracts from cells harboring pET-3a/MJ0430, whereas extracts of strains containing pET-3a or pET-3a/MJ1102 did not result in any change of the dCTP spectrum. None of the extracts catalyzed the deamination of dCMP, as measured spectrophotometrically. Thus, it was concluded that the gene product of MJ0430 was a dCTP deaminase/dUTPase and that of MJ1102 was a dUTPase.
Molecular Properties of Purified dCTP DeaminaseThe dCTP deaminase was purified to homogeneity in three steps: heat treatment, chromatography on Q-Sepharose, and finally chromatography on Red-Sepharose as described under "Experimental Procedures." Phenylmethylsulfonyl fluoride and EDTA were included in all steps to avoid an apparently specific degradation reducing the size of the polypeptide chain from 28 kDa stepwise toward an end product of 26 kDa. According to matrix-assisted laser desorption/ionization mass spectrometry, the mass of the purified enzyme was 23,331 Da, whereas the predicted mass calculated from the DNA sequence was 23,432 Da. These two figures, differing by 101 Da, are in reasonable agreement because the experimental error from the instrument is between 0.1 and 0.5%. The proteolytically degraded form of the enzyme from an early purification attempt was stable toward further proteolysis and lacked both dCTP deaminase and dUTPase activity. Its mass of 21,212 Da from matrix-assisted laser desorption/ionization mass spectrometry suggested a loss of 1520 residues. The N terminus of the degraded form was intact according to Edman degradation (MILSDKDIID), indicating that the cleavage site(s) mapped to the C terminus. This may indicate that the C-terminal residues are located on a flexible part of the molecule, a feature previously observed for trimeric dUTPases (25, 26, 27).
Reaction Products from [3H]dCTPAs mentioned above, assays of permeabilized cells demonstrated that the MJ0430 protein catalyzed deamination of dCTP, but the spectral analyses did not provide identification of the reaction product. The activity of purified MJ0430 protein was analyzed with dCTP deaminase Assay 1 in which the separation on TLC was designed to identify the possible reaction products from [3H]dCTP. Throughout the reaction, all label that disappeared from dCTP was found in dUMP. No radioactivity was detected in dUTP or dCMP. Thus, dUTP or dCMP did not accumulate as intermediates of the reaction. In a parallel experiment, the reaction with [3H]dCTP (1 mM) was carried out in the presence of unlabeled dUTP (1 mM) (Fig. 2). The rate of the reaction (disappearance of dCTP) was lower (0.2 compared with 0.3 s1), because of inhibition by dUTP, but again no label was found in the spots corresponding to dUTP and dCMP. Throughout the reaction, all counts disappearing from dCTP could be accounted for in dUMP (Fig. 2). This showed that the presumed first reaction product from dCTP deamination, i.e. dUTP, did not equilibrate with external dUTP. It cannot be ruled out that both chemical transformations occur in a concerted reaction without dUTP as an intermediate. However, because dUTP was accepted as a substrate with high turnover, this seems less likely.
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Kinetics of the dCTP Deaminase ReactionThe deamination reaction showed an absolute dependence on Mg2+ activity (data not shown), so Mg2+ was present in excess in all assays. A progress curve of the deamination of 2 mM dCTP is shown in Fig. 3. The extended linear phase (015 min), representing saturation with substrate, was used to calculate a turnover number of 0.3 s1 at 25 °C. At 75 °C, a similar assay indicated a rate of 5 s1, i.e. almost 20-fold higher. Assay 3 was employed to make initial rate measurements over a range of dCTP concentrations. The sigmoid shape of the saturation curve indicated that the enzyme did not follow Michaelis-Menten kinetics under these conditions (Fig. 4). Half saturation (S0.5) was obtained at about 40 µM dCTP, and the Hill coefficient was 1.9, suggesting positive cooperativity. Inhibition by dTTP and dUTP was measured over the same range of dCTP concentrations. They both inhibited the deamination reaction, but neither the Hill coefficient nor the maximal velocity was significantly affected, indicating simple competitive inhibition with apparent Ki values of 0.1 and 0.5 mM, respectively. The relatively high affinity for dTTP may be physiologically relevant for regulation of enzyme activity.
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The deamination of a few (deoxy)cytidine compounds (at 200 µM) was tested using Assay 3. Less than 0.5% activity was obtained with dCMP, dCDP, and CTP, whereas ddCTP was deaminated at the same rate as dCTP.
Kinetics of the dUTPase ReactionAs shown in Table I, the MJ0430 and the MJ1102 proteins catalyzed the conversion of dUTP to dUMP with about equal efficiency at a dUTP concentration of 0.9 mM. However, at a dUTP concentration of 10 µM, large differences between the two enzymes became apparent (Fig. 5). With the MJ0430 protein, no linear phase was observed, indicating that the enzyme was far from being saturated. Measurements using higher dUTP concentrations in the presence of similarly increased buffer capacity indicated a Km value in the mM range. For the MJ1102 protein, the linear phase lasted until a very low substrate concentration was reached, and a Km value of 0.4 µM was calculated. This is within the range for other dUTPases characterized by the same method, e.g. 0.2 µM for the E. coli enzyme (9) and 1.1 µM for the equine infectious anemia virus dUTPase (10).
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DISCUSSION |
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The ability of the bifunctional enzyme to release dUMP and not dUTP as the product of the deamination seems advantageous because of the hazards of uracil incorporation. Uracil in DNA may also arise by spontaneous deamination of cytosine residues (29). Because the repair process may cause DNA fragmentation (21), the involvement of dUTP as an intermediate in the metabolic pathway to dTTP appears dangerous. It is limited, however, to microorganisms because eukaryotes carry out the deamination at the monophosphate level. Among the Archaea it appears that either a dCTP deaminase or a dCMP deaminase is present. Annotations of dCMP deaminases in the data bases, e.g. for Archeoglobus fulgidus and Pyrococci, seem unambiguous because of the presence of a highly conserved sequence within this family of enzymes (30). It includes the zinc-coordinating residues and a glutamate residue involved in acid/base catalysis. This conserved sequence is not found in dCTP deaminases. dUTPases are also required in cells that depend on dCMP deamination for generating dUMP because there are other sources of dUTP: (i) reduction of UDP (UTP) by ribonucleotide reductase and (ii) sequential phosphorylations of dUMP and dUDP by thymidylate kinase and nucleoside diphosphate kinase (1).
The properties of the two M. jannaschii proteins described in this report, i.e. the lack of release of the intermediate dUTP from the bifunctional MJ0430 protein and the high affinity for dUTP of the MJ1102 protein, are consistent with the importance of keeping the intracellular concentration of dUTP very low. The observation that the activity of certain archaeal DNA polymerases, in contrast to eubacterial and human DNA polymerases, was inhibited by uracil-containing oligonucleotides (31) suggests that the cellular defense against uracil incorporation may also involve the DNA polymerases in Archaea.
The bifunctional M. jannaschii enzyme is not a fusion protein of a dCTP deaminase and a dUTPase. Its polypeptide chain is only 11 residues longer than the 193 residues of the monofunctional E. coli dCTP deaminase. The relationship between dCTP deaminase and dUTPase is interesting in terms of evolution. Three of the five conserved motifs in dUTPase, motifs 2, 3, and 4, are discernible in the dCTP deaminases, and an alignment of these sequences with dUTPase displays no large gaps or inserts (12), indicating common structural features. Motif 3 has been suggested as an ancient structure for pyrimidine binding (6, 11). In dUTPase it has also a role in recognition of the pentose moiety of the substrate (5, 6, 7, 8), which may also be the case for dCTP deaminase. The residues immediately before and after the core of motif 3 may have different roles. In the dUTPases, these residues are involved in substrate discrimination, i.e. excluding the pyrimidine rings of dCTP and dTTP from the active site. However, most questions regarding the structural relationship between dCTP deaminases and dUTPases, such as quartenary structure and communication between the subunits upon ligand binding, have to await the first three-dimensional structure of a dCTP deaminase. Such a structure of the bifunctional MJ0430 protein would help to reveal how the two reactions catalyzed by the enzyme are carried out without release of the putative intermediate of the reaction, dUTP.
P. furiosis dUTPase is added to commercial preparations of thermostable DNA polymerases as an enhancing factor (Pfu Turbo DNA polymerase) (13). By scavenging dUTP from PCR mixtures, the thermostable enzyme enables synthesis of long DNA fragments (>10 kilobase pairs). Archaeal dUTPases, such as the P. furiosis and M. jannaschii enzymes, are worth attention not only for biotechnological applications. Compared with the eubacterial and eukaryotic dUTPases, the archaeal dUTPases lack the typical glycine-rich motif 5, whose residues at the C termini are known to be highly flexible, as shown by trypsinolysis and NMR studies (25, 27), and usually not seen in the x-ray structures. The essential role of motif 5 has been confirmed by site-directed mutagenesis (26). The absence of this motif may be an environmental adaptation to high temperatures, and it is certainly a major reason for the previous incorrect annotations of archaeal dUTPases as dCTP deaminases. As noticed by Prangishvili et al. (12), a BLAST search of the SIRV dUTPase indicated a closer match to E. coli dCTP deaminase than to E. coli dUTPase. However, the three identified archaeal dUTPases, i.e. from SIRV, P. furiosis, and here M. jannaschii, contain a short sequence (YXGXY), close to their C termini, which may be regarded as archaeal motif 5.
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FOOTNOTES |
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|| To whom correspondence should be addressed. Tel.: 45-35322002; Fax: 45-35322040; E-mail: neuhard{at}mermaid.molbio.ku.dk.
1 J. Neuhard, unpublished results.
2 The abbreviation used is: Bicine, N,N-bis(2-hydroxyethyl)glycine.
3 J. Nord, personal communication.
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ACKNOWLEDGMENTS |
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REFERENCES |
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