Archaebacterial DNA polymerases tightly bind uracil-containing DNA.

We show that archaebacterial DNA polymerases are strongly inhibited by the presence of small amounts of uracil-containing DNA. Inhibition appears to be competitive, with the DNA polymerase exhibiting ∼6500-fold greater affinity for binding the inhibitor than a DNase I-activated DNA substrate. All six archaebacterial DNA polymerases tested were inhibited, while no eubacterial, eukaryotic, or bacteriophage enzymes showed this effect. Only a small inhibition resulted when uracil was present as the deoxynucleoside triphosphate, dUTP. The rate of DNA synthesis was reduced by ∼40% when dUTP was used in place of dTTP for archaebacterial DNA polymerases. Furthermore, an incorporated dUMP served as a productive 3′-primer terminus for subsequent elongation. In contrast, the presence of an oligonucleotide containing as little as a single dUrd residue was extremely inhibitory to DNA polymerase activity on other primer-template DNA.

During the last few years, several DNA polymerases have been identified from thermophilic archaebacteria (1)(2)(3)(4). The small number of representatives available thus far have shared sequence similarities (5), and the purified enzymes have had several notable properties in common. All are thermostable as anticipated. Each is associated with a 3Ј35Ј proofreading exonuclease activity, and none have a 5Ј33Ј exonuclease activity. Archaea is a third kingdom distinct from eubacteria and eukaryotes (6,7) and is thought to be evolutionarily closer to the eukaryotes. Some archaebacterial proteins share strong homology with eukaryotic counterparts such as RNA polymerase; however, some transcription-associated genes are organized in clusters resembling those of eubacteria (8). The archaebacterial DNA polymerases share significant homology with the family B DNA polymerases (5), which include eukaryotic cell DNA polymerases as well as Escherichia coli pol 1 II and bacteriophage T4 DNA polymerase.
We report here an unusual property that is apparently unique to the archaea. The presence of uracil in DNA results in a dramatic increase in the binding affinity of the DNA polymerase. We first observed this effect while attempting to carry out polymerase chain reaction protocols that included dUrdcontaining oligonucleotides. These oligonucleotides appeared to block all DNA synthesis, suggesting a direct action on the DNA polymerase. Evidence is presented that suggests that the DNA polymerase forms a tight nonproductive complex with dUrd-containing DNA. We speculate on the possibility that this effect is related in some way to the extreme temperatures at which these thermophiles live and that it may serve in a biological function.

MATERIALS AND METHODS
Oligonucleotides-Oligonucleotides were obtained from Life Technologies, Inc. ( Table I). All of the oligonucleotides were determined to be substantially free of secondary structure by analysis with the Oligo Primer Analysis Program, Version 5.0 for Windows (National Biosciences, Inc., Plymouth, MN).
Enzymes-Recombinant Thermus aquaticus (Taq) DNA polymerase, E. coli DNA pol I, and T4 DNA polymerase were purchased from Life Technologies, Inc. DNA polymerases from bacteriophage T5 (9), Desulfurococcus strain Tok12-S1 (Dtok), 2 Thermotoga neapolitana (Tne), 2 and Thermus thermophilus (Tth) 3 1 The abbreviations used are: pol, polymerase; UDG, uracil DNA glycosylase. measure inhibition of DNA synthesis, DNA polymerases were preincubated with inhibitors or controls (Table I) for 2 min at the reaction temperature to allow binding of the polymerase to come to equilibrium, and then reactions were started by the addition of dNTPs and activated DNA.
UDG Treatment of Oligonucleotides-145 pmol of the dUrd-85-mer were incubated at 37°C for 15 min in the presence or absence of 1 unit of UDG (Life Technologies, Inc.) in a 29-l reaction containing 20 mM Tris-HCl (pH 8.5) and 50 mM KCl. Then 5 pmol of the oligonucleotide were added to 200 l of Vent exo (Ϫ) DNA polymerase reactions containing 100 g/ml activated salmon testis DNA substrate as described under "Assays for DNA Polymerase Activity and Inhibition." At the time points indicated, 20-l aliquots of the reaction mixture were added to 5 l of 500 mM EDTA.
Kinetic Gel Assay for dUTP Incorporation-The assay was carried out essentially as described (10,11). The primer-template was designed with a single template A 16 nucleotides from the 3Ј-primer terminus ( Table I). The primer and template oligonucleotides were purified by polyacrylamide gel electrophoresis. The primer was end-labeled using [␥-32 P]ATP (3000 Ci/mmol; Amersham Corp.) and polynucleotide kinase (Life Technologies, Inc.), annealed to a 2-fold excess of template by heating to 90°C for 2 min, and then left at room temperature for 1 h. The primer-template was separated from unutilized [␥-32 P]ATP with a 1.5-ml Sephadex G-25 column. 20-l primer extension reactions contained a 90 nM final concentration of primer-template, Vent reaction buffer (see "Assays for DNA Polymerase Activity and Inhibition"); 200 M concentrations each of dATP, dCTP, and dGTP (Life Technologies, Inc.); and either dUTP (Pharmacia) or dTTP at concentrations ranging from 0 to 20 M and 0.02 unit of Vent exo(Ϫ) DNA polymerase. Reactions were incubated for 5 min at 70°C in a Perkin-Elmer 9600 thermocycler. Prior experiments demonstrated that these conditions would extend Ͻ10% of the primers as required for the analysis of kinetic parameters (12). Reactions were stopped by addition of 100 l of 32% (v/v) formamide and 17 mM EDTA. This large volume of stop solution gave a 6-fold dilution of the reaction that seemed to improve resolution by electrophoresis; 3 l were resolved on an 8% polyacrylamide sequencing gel. The gels were analyzed with a Molecular Dynamics Phos-phorImager using MultiQuant software version 3.3. Kinetic parameters were calculated as described (11).
Determination of K I for Uracil-containing DNA-200-l reactions were carried out as described above for DNA polymerase inhibition except that 2 units of Vent exo (Ϫ) DNA polymerase were preincubated for 3 min at 70°C with 75, 300, 600, or 1500 M (nucleotide) DNase I-activated salmon testis DNA in the presence or absence of 2.125 M (nucleotide) dUrd-85-mer, and then the reaction was started by adding dNTPs. Aliquots were removed for assaying at different times. Plots of the reaction time courses were analyzed to determine initial rates, and the data were replotted in a Lineweaver-Burk plot. The K I was derived from the relationship: slope in the presence of inhibitor ϭ K m /V max (1 ϩ [I]/K I ) (13).

Inhibition of Archaebacterial DNA Polymerase Activity by a
Uracil-containing Oligonucleotide-In the course of studies using dUrd-containing primers as a method for cloning polymerase chain reaction amplified DNA (14, 15), we observed that when archaebacterial DNA polymerases were used, reactions were consistently inhibited. Using an 85-base oligonucleotide containing dUrd (dUrd-85-mer, Table I) we studied the effect of dUrd-containing DNA on archaebacterial DNA polymerases.
The dUrd-85-mer inhibited Vent exo (Ϫ) DNA polymerase from utilizing a primed M13 single-stranded DNA substrate (Fig. 1). From 2 to 5 pmol of the dUrd-85-mer effectively blocked synthesis by ϳ2 pmol of DNA polymerase, suggesting exceptionally tight binding of the enzyme to uracil-containing DNA. A control 85-mer with the same sequence but containing dThd instead of dUrd (dThd-85-mer, Table I) did not block the reaction. The dUrd-85-mer gave a similar level of inhibition to Vent exo (ϩ) DNA polymerase, indicating that the 3Ј35Ј exonuclease activity of the enzyme does not effect the level of inhibition (data not shown). With DNase I-activated salmon testis DNA as the substrate, 16 other DNA polymerases were tested for inhibition by the uracil-containing 85-mer (Table II). All five of the archaebacterial DNA polymerases were inhibited at levels similar to those of Vent DNA polymerase. None of the other DNA polymerases was inhibited including five thermostable eubacterial enzymes derived from Thermus and Thermotoga spp. Human DNA pol ␣, E. coli pol II, and bacteriophage T4 DNA polymerase were not inhibited in spite of sharing sequence similarities with the archaebacterial enzymes (5). A 42-mer with a sequence unrelated to the dUrd-85-mer and containing 10 dUrd residues (dUrd-42-mer, Table I) was also strongly inhibitory, suggesting that the presence of dUrd in any DNA sequence is sufficient for inhibition. A 71-mer containing a single dUrd substituted for thymine at position 23 (single dUrd-71-mer, Table I) was also strongly inhibitory for Vent exo (Ϫ) DNA polymerase. The single dUrd-71-mer inhibited 60% of the activity, while the dUrd-85-mer (which has 22 dUrd residues; see Table I) inhibited 94% of the activity. Globin mRNA had no inhibitory effect. Therefore, uracil in RNA does not have the same effect as dUrd in DNA. Inhibition from the dUrd-85-mer was not reduced when it was annealed with a complementary 85-mer (not containing dUrd) (Fig. 2). In this experiment, the dUrd-85-mer was annealed to an 8-fold excess of the complementary 85-mer before use as an inhibitor to DNA synthesis. The double-and single-stranded forms of the dUrd-85-mer had nearly identical levels of inhibition.
Relief of Inhibition by Treatment with Uracil DNA Glycosylase-As a further control to demonstrate the direct role of uracil in inhibition, the dUrd-85-mer was pretreated with UDG for removal of uracil bases from DNA. Pretreatment with UDG acted to relieve the inhibitory effect of the dUrd-85-mer (Fig. 3). UDG pretreatment of the dThd-85-mer control oligonucleotide (which is not an inhibitor; see Fig. 1) had no effect on the rate of DNA synthesis (data not shown).
Competitive Inhibition of DNA Polymerase by a Uracil-containing Oligonucleotide-To estimate the effect of uracil on Vent exo (Ϫ) DNA polymerase, the K m app was measured for DNase 1 activated DNA substrate in the presence or absence of a fixed amount of dUrd-85-mer (Fig. 4). The increase in the slope for the Lineweaver-Burk plot in the presence of inhibitor is characteristic of competitive inhibition and indicates that the

5Ј-UCAUCGAGCAUGAUCAGGUCGUGACUGGGACGCCAUGUCUGC-3Ј
Primer-template for kinetic gel assay K I for the dUrd-85-mer is about 6500-fold lower than the K m app for the activated DNA substrate (see "Materials and Methods").
Utilization of dUTP by Vent DNA Polymerase-A kinetic gel assay (10, 11) was used to determine whether uracil was inhibitory when it was present as the deoxynucleoside triphosphate, dUTP. This experiment allowed the direct measurement of the initial rate of incorporation as distinguished from any subsequent inhibition from the nascent dUrd-containing product which was predicted to be an inhibitor just like the dUrd-85-mer. Vent DNA polymerase was used because Vent is a well characterized representative of archaebacterial DNA polymerases (16), and the exo (Ϫ) form was used because enzymes lacking a 3Ј35Ј exonuclease activity require a simpler analysis (12). A primer-template (Table I) was designed with a single template A target site, designated position N, 16 nucleotides downstream from the 3Ј-primer terminus. In a reaction containing dATP, dCTP, and dGTP but lacking dTTP, the DNA polymerase rapidly extended the primer by 15 nucleotides but was strongly blocked at the target A. The result is a strong band on a DNA sequencing gel one nucleotide before the target A at position N Ϫ1 (Fig. 5A, arrow). Only a small proportion of the primers were extended past the A due to misincorporated dAMP, dCMP, and dGMP (12). The addition of dTTP or dUTP relieved the block as indicated by the loss of the band at N Ϫ1 and the appearance of longer products. The relative rate for dUMP and dTMP incorporation was determined by comparing the concentration of nucleotide required to relieve the block. The relative velocity of incorporation is expressed as the ratio   of the summed band intensities for position ՆN divided by the band intensity at N Ϫ1 (10, 11) (Fig. 5B). dUMP had an incorporation rate characteristic of a normal nucleotide, being only about 40% less than for dTMP. Therefore, the extreme inhibition from uracil-containing DNA (Fig. 1) is not observed when uracil is present as a deoxynucleoside triphosphate.
The DNA polymerase is also not inhibited immediately after incorporation of dUMP onto the primer terminus, as indicated by the rapid extension of primers past the target A site. If dUMP had been incorporated rapidly but then served as a poor substrate for subsequent extension, this would be revealed by the accumulation of product at the target position N or other downstream positions. No accumulation of these products is observed (Fig. 5A). As a further demonstration of this point, a time course of the reaction was carried out with saturating dUTP or dTTP, and the accumulation of full-length runoff products was measured. The full-length products accumulated at the same rate whether dUTP or dTTP was used (Fig. 6). This result requires that dUMP be rapidly incorporated and that subsequent extension of the primer is not blocked. Therefore, uracil at a 3Ј terminus does not trap the DNA polymerases in the same way that the uracil-containing 85-mer did (Fig. 1). To be certain that uracil was really the predominant base incorporated at the target A site, some of the runoff products were treated with UDG. More than 90% of these were cut (data not shown).

DISCUSSION
The goal of this study was to identify the nature of the inhibitory effect of uracil-containing DNA and the extent of the effect among DNA polymerases from different organisms. Inhibition of DNA polymerase apparently resulted from an extremely tight, nonproductive binding of the inhibitor. A kinetic analysis indicated a ϳ6500-fold greater affinity of the DNA polymerase for the inhibitor over the activated DNA substrate (Fig. 4). Levels of activated DNA and the dUrd-85-mer were expressed as concentrations of nucleotides. No attempt was made to control for the difference in the length of the inhibitor (85 nucleotides) and the gapped DNA substrate (average length, several hundred nucleotides). While the measured K I is specific for this inhibitor, other uracil containing oligonucleotides (Table I) and also an alternative DNA substrate, primed M13 single-stranded DNA, suggested K I values of similar mag-nitude. Although every dUrd-containing oligonucleotide tested was inhibitory, the K I may prove to be influenced by local sequence context as well as oligonucleotide length. The percentage of inhibition for a given concentration of inhibitor decreased with increasing activated DNA substrate consistent with a simple model of competitive inhibition. The extreme preference for binding inhibitor is consistent with the close stoichiometry between inhibitory levels of the dUrd-85-mer and DNA polymerase. Approximately 2 pmol of Vent exo (Ϫ) DNA polymerase were almost completely inhibited by 2-5 pmol of the dUrd-85-mer (Fig. 1). The direct role of uracil in the binding was shown by the relief of inhibition by treatment with FIG. 4. Effect of the dUrd-85-mer on Vent exo (؊) DNA polymerase's apparent K m for the DNA substrate. Initial rates of DNA synthesis were determined for various concentrations of the DNase I-activated salmon testis DNA substrate and then plotted above as described under "Materials and Methods." The dUrd-85-mer was either present (q) or absent (E). In the absence of the dUrd-85-mer, the calculated V max was 0.6 pmol/min, and the apparent K m was 380 M (nucleotide).

FIG. 5. Incorporation kinetics of dUTP compared with dTTP.
At the dUTP or dTTP concentrations indicated, 5-min reactions were carried out and resolved on a denaturing polyacrylamide sequencing gel as described under "Materials and Methods." The 5Ј-32 P-labeled primer was annealed to a template with a single A residue 16 nucleotides downstream from the primer terminus (referred to as position N). A, at limiting concentrations of dUTP and dTTP, a dark band appears one nucleotide before position N, at position N Ϫ1 (arrow). The calculated relative velocity of incorporation of dUTP (q) and dTTP (E) are also plotted (B). UDG (Fig. 3). Furthermore, an oligonucleotide of the same sequence but containing dThd in place of dUrd was not inhibitory. Even the presence of a single uracil in an oligonucleotide was sufficient for severe inhibition (Table II). Inhibition was about the same for double-and single-stranded dUrd-85-mer DNA (Fig. 2). Globin mRNA had no inhibitory effect. Therefore, uracil in RNA does not have the same effect as dUrd in DNA, although this may simply reflect differences in DNA polymerase affinity between RNA and DNA in general.
We also investigated the effect of uracil in the deoxynucleoside triphosphate, dUTP. A previous study (17) concluded that for Vent and Pfu DNA polymerases, dUTP was a poor substrate for PCR polymerase chain reaction and also primer extension in general. Use of the kinetic gel assay (10, 11) allowed us to measure the true polymerization rate for a single dUMP residue at a specific site. This assay is independent of DNA polymerase concentration and should also be independent of any sequestration of polymerase by accumulating uracil containing DNA product. dUTP was utilized fairly normally as a substrate for the Vent exo (Ϫ) DNA polymerase, with a rate ϳ60% of that for dTTP (Fig. 5). Further extension of the primer downstream also occurred at the same rate whether dUrd or dThd was incorporated at the target site (Fig. 6). Therefore, uracil that has just been inserted during ongoing DNA synthesis does not seem to pose the same severe inhibition as seen when an oligonucleotide containing dUrd is presented as an inhibitor to polymerase binding of another DNA substrate. It can also be inferred from these experiments that the presence of dUTP in the reaction does not inhibit incorporation of the normal dNTPs in contrast to the inhibition from the dUrd-containing oligonucleotides. It is likely that the oligonucleotides require some minimal length before the presence of dUrd causes inhibitory binding.
The extremely strong effect of uracil demonstrated here is even more remarkable in that it is seen only for this specific group of DNA polymerases. All archaebacterial DNA polymerases tested were inhibited at similar levels. To date, the number of DNA polymerases available from archaebacteria is still small; and even though three genera of archaea were represented in this study, they may actually represent a fairly small subset of closely related species. The identification of new polymerases will allow a further investigation of how widespread the characteristic is. No eubacterial, eukaryotic, or bacteriophage DNA polymerases were inhibited. Human pol ␣, E. coli pol II, and bacteriophage T4 DNA polymerase were of particular interest because they are reportedly related to the Archaea polymerases (5). However, no inhibition was detected. The inhibitory effect is not entirely correlated with the thermostability of the DNA polymerases, since the thermophilic eubacterial enzymes derived from Thermus and Thermotoga spp. were not inhibited. However, we do note that the archaebacteria involved can generally live at higher temperatures than Thermus and Thermotoga spp. and may be adapted for more extreme thermostability. Furthermore, it remains possible that the inhibition observed relates to some adaptation involving life at extreme temperatures that is absent in the evolutionarily divergent eubacteria. One possibility is that the strong binding serves a biological role involving the recognition and repair of uracils in vivo. In E. coli, uracil glycosylase recognizes uracils in DNA and excises them (18). Further research on archaebacteria may reveal whether the binding observed here is involved in this type of repair system. We have searched for primary amino acid sequence homology or conserved motifs in the sequences of known DNA glycosylases and archaebacterial DNA polymerases. There are no obvious conserved motifs in the primary sequence. Although the crystal structure of UDG is known (19,20), no archaebacterial DNA polymerase structure is available yet.
FIG. 6. Effect of dUTP or dTTP on accumulation of full-length extended primers. Reactions were the same as in Fig. 5 except that a time course was carried out and the accumulation of full-length runoff products was determined as a measurement of the incorporation and subsequent elongation for saturating dUTP (q) or dTTP (E).