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J. Biol. Chem., Vol. 275, Issue 25, 19146-19149, June 23, 2000

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Uracil-DNA Glycosylase in the Extreme Thermophile Archaeoglobus fulgidus^*

Margarita Sandigursky and William A. Franklin

From the Departments of Radiology and Radiation Oncology, Albert Einstein College of Medicine, Bronx, New York 10461

Received for publication, March 9, 2000, and in revised form, April 14, 2000

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Uracil-DNA glycosylase (UDG) is an essential enzyme for maintaining genomic integrity. Here we describe a UDG from the extreme thermophile Archaeoglobus fulgidus. The enzyme is a member of a new class of enzymes found in prokaryotes that is distinct from the UDG enzyme found in Escherichia coli, eukaryotes, and DNA-containing viruses. The A. fulgidus UDG is extremely thermostable, maintaining full activity after heating for 1.5 h at 95 °C. The protein is capable of removing uracil from double-stranded DNA containing either a U/A or U/G base pair as well as from single-stranded DNA. This enzyme is product-inhibited by both uracil and apurinic/apyrimidinic sites. The A. fulgidus UDG has a high degree of similarity at the primary amino acid sequence level to the enzyme found in Thermotoga maritima, a thermophilic eubacteria, and suggests a conserved mechanism of UDG-initiated base excision repair in archaea and thermophilic eubacteria.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Uracil-DNA glycosylase (UDG)¹ is a ubiquitous enzyme found in most eukaryotes and prokaryotes (1-3). This enzyme removes uracil that is present in DNA either due to deamination of cytosine or misincorporation of dUMP in place of dTMP (4, 5) and is the primary activity in the base excision repair pathway for the removal of uracil from DNA. The protein has been well characterized in both Escherichia coli and from eukaryotic cells; the crystal structures of the E. coli, human, and herpes simplex virus UDGs have been solved (6-8). A high degree of similarity has been noted for the E. coli enzyme and its eukaryotic analogues; for example, the human enzyme and the E. coli proteins are 55.7% identical (9).

UDG activities have been shown to be present in several thermophiles (10-12). However, several bacterial genomes lack sequences complementary to the E. coli ung gene (13). This suggests that if UDG activities are present in these organisms, they may differ significantly from the E. coli/eukaryotic/viral UDG enzymes at least at the primary amino acid sequence level.

We have isolated a gene from the thermophile Thermotoga maritima that expresses a uracil-DNA glycosylase (14). The gene was discovered by having weak sequence similarity to the E. coli G:T/U mismatch-specific DNA glycosylase (mug) gene. The protein is thermostable and acts to remove uracil from both U/A and U/G base pairs in DNA. Analogous genes appear to be present in several other prokaryotic organisms in both eubacteria and archaea. These findings suggest that the T. maritima UDG is a member of a new class of DNA repair enzymes.

In this study we describe the isolation and characterization of the uracil-DNA glycosylase from Archaeoglobus fulgidus (15). This is the first UDG to be isolated from archaea. This protein is highly homologous to the enzyme from T. maritima, yet is considerably more heat-stable. These findings suggest a conserved mechanism of uracil base excision repair in archaea.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Bacterial Strains and Plasmids-- BW310 (l-, ung-1, relA1, spoT1, thi-, obtained from E. coli Genetic Stock Center, Yale University) was lysogenized with DE3 using the  lysogenation kit from Novagen. The plasmid pET28a was obtained from Novagen.

Cloning of the A. fulgidus UDG Gene-- PCR was carried out using a pUC18 plasmid containing an insert of A. fulgidus genomic DNA (GAFFT53 pUC18 TIGR clone, obtained from American Type Culture Collection) as template, and the oligonucleotides 5'-GGGGAAGCTAGCATGGAGTCTCTGGACGAC-3' and 5'-GGCCGGGGATCCTCATAGGTAATCAAAGAG-3' containing NheI and BamHI restriction sites at the 3' and 5' ends, respectively, for subsequent cloning into the pET28a vector system (Novagen). The DNA sequence of the insert was confirmed by DNA sequencing analysis. The plasmid expressing the His tag fusion protein, pET28a-afung, was expressed in E. coli strain BW310(DE3).

Enzyme Purification-- BW310 (pET28a-afung) was inoculated into LB medium containing 34 mg/ml kanamycin (LB-kan) and was grown overnight at 37 °C. The overnight culture was diluted 1:50 with fresh LB-kan medium and was grown at 37 °C until the A₆₀₀ of the culture reached 0.8. Isopropyl-1-thio--D-galactopyranoside was then added to a final concentration of 1 mM, and the culture was incubated for an additional 3 h at 30 °C. Cells were pelleted by centrifugation at 3,000 × g for 5 min at 4 °C and then resuspended in 2 ml of ice-cold buffer containing 5 mM imidazole, 500 mM NaCl, and 20 mM Tris-HCl, pH 7.9 (1× binding buffer). Cells were lysed by sonication with 4 × 10-s bursts. The sonicate was clarified by centrifugation at 12,000 × g at 4 °C for 30 min (fraction I). Fraction I (3 ml, 2 mg/ml) was applied at a flow rate of 0.5 ml/min to a 1.2-ml His-Bind Resin Ni²⁺ column (Novagen), which was subsequently washed with 12 ml of 1× binding buffer. Protein was eluted from the column with buffer containing 60 (6 ml), 100, 250, and 500 mM (3 ml each) imidazole in 500 mM NaCl, 20 mM Tris-HCl, pH 7.9. AFUDG was mainly found in the 60 mM imidazole fraction (fraction II). Fraction II (2.5 ml, 80 µg/ml) was loaded on a PD-10 gel filtration column (Amersham Pharmacia Biotech) and eluted with 3.5 ml of buffer A (50 mM Hepes-KOH, pH 7.8, 0.1 mM EDTA, 1 mM DTT, 5% glycerol) (fraction III). Fraction III (3 ml, 55 µg/ml) was applied to a MonoS HR 5/5 column (Amersham Pharmacia Biotech), and protein was eluted from the column with a 20-ml linear gradient from buffer A to buffer A containing 1 M NaCl at a flow rate of 1 ml/min. Fractions (0.5 ml each) were assayed for AFUDG activity. Active fractions were pooled (fraction IV). The enzyme was eluted with a salt concentration of 0.45-0.5 M NaCl. Fraction IV (1.0 ml, 85 µg/ml) was added to an equal amount of glycerol and was stored at 20 °C.

DNA Substrates-- DNA containing ³H-labeled uracil was prepared by nick translation of calf thymus DNA as described previously (14). Oligonucleotide substrates were prepared as follows: 30-mer 5'-ATATACCGCGG(U/C)GGCCGATCAAGCTTATT-3' was 5'-end-labeled with ³²P and was annealed to either 5'-AATAAGCTTGATCGGCCGACCGCGGTATAT-3' to give a double-stranded 30-mer with a single U/A base pair or to 5'-AATAAGCTTGATCGGCCGGCCGCGGTATAT-3' to give a double-stranded 30-mer with a single U/G base pair. An analogous substrate containing a T/G base pair was also prepared. The annealing of the oligonucleotides was performed as described previously (14, 16). Double-stranded oligonucleotides containing AP sites were prepared as follows: unlabeled double-stranded 30-mers (15 nmol) were incubated with 150 ng of AFUDG at 37 °C overnight (16 h) in 50 mM MOPS-KOH, pH 7.8, 0.1 mM EDTA, 1 mM DTT, 100 µg/ml BSA (Promega; nuclease and uracil-DNA glycosylase-free) in a total volume of 200 µl. Following the reaction, an equal volume of phenol/chloroform was added to the reaction mixture, and the oligonucleotides containing AP sites were recovered following ethanol precipitation and lyophilization and were dissolved in 150 µl of 10 mM Tris-HCl, pH 7.8, 1 mM EDTA.

Reactions with Double-stranded DNA-- Reactions (100 µl) contained 0.75 pmol of DNA substrate containing ³H-labeled uracil (15,000 cpm), 50 mM MOPS-KOH, pH 7.8, 0.1 mM EDTA, 1 mM DTT, 100 µg/ml BSA, 0.1 pmol of AFUDG protein and were incubated at 70 °C for 10 min. Reactions were stopped by the addition of 110 µl of 10% trichloroacetic acid and 11 µl of calf thymus DNA (2.5 mg/ml). The samples were centrifuged at 10,000 × g for 5 min. Radioactivity contained in the supernatant was determined by liquid scintillation counting.

Reactions with Single-stranded DNA-- A solution (100 µl) containing 0.75 pmol of DNA substrate containing ³H-labeled uracil (15,000 cpm), 50 mM MOPS-KOH, pH 7.8, 0.1 mM EDTA, 1 mM DTT, 100 µg/ml BSA was incubated at 95 °C for 10 min. AFUDG (0.1 pmol, preincubated at 95 °C) was added, and the reaction was continued for 10 min. Reactions were stopped by the addition of 110 µl of 10% trichloroacetic acid and 11 µl of calf thymus DNA (2.5 mg/ml). The samples were centrifuged at 10,000 × g for 5 min. Radioactivity contained in the supernatant was determined by liquid scintillation counting.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A. fulgidus Uracil-DNA Glycosylase-- An open reading frame (ORF) analogous to the UDG gene from T. maritima (14) was identified following a BLAST (17) search of the A. fulgidus genomic DNA (15). This ORF was identified at the Institute for Genomic Research data base (locus AF2277) as being homologous to a DNA polymerase from the Bacillus subtilis bacteriophage SPO1 (18). This ORF encodes a 199-amino acid protein of 22,718 daltons and has a pI of 6.75. The sequence of this ORF was amplified by PCR, and the PCR product was cloned into an expression vector, pET28a, which places a histidine tag at the 5' end of the gene. The gene was expressed in an E. coli strain deficient in UDG activity, and the expression product was purified as a His tag fusion protein as shown in Fig. 1.

View larger version (92K): [in this window] [in a new window]   Fig. 1.   Purification of A. fulgidus UDG. The purity of the enzyme was evaluated on a 12% SDS-polyacrylamide gel that was stained with Coomassie Blue. Lanes 1 and 5, molecular weight markers; lane 2, fraction I (6 µg); lane 3, fraction II (2 µg); lane 4, fraction IV (2.2 µg). The sizes of the molecular mass markers are given in the margin in kDa. It is estimated that purity of the protein in fraction IV is >95%.

Activity on Double-stranded DNA-- The UDG activity of the expressed protein was determined using a double-stranded DNA substrate containing ³H-labeled uracil substituted for thymine and was measured at 70 °C. The protein did not lose activity when preincubated without substrate at 95 °C for up to 1.5 h. The enzyme was also active at temperatures 37 °C and above. A time course for the release of uracil at 70 °C is shown in Fig. 2. The K_m for release of uracil from this substrate was determined from Lineweaver-Burk analysis to be 0.5 µM, over a substrate range of 0.03 to 0.6 µM (Fig. 3). The enzyme did not contain any apurinic/apyrimidinic endonuclease or lyase activities, as well as exonuclease activities, and did not function as a DNA polymerase. The enzyme demonstrated no difference in activity within a pH range of 7.0 to 8.5. We have denoted the enzyme as A. fulgidus UDG (AFUDG); the gene is denoted as afung.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   Time course for the release of uracil from a double-stranded DNA substrate containing 3H-labeled uracil. Reactions were incubated at 70 °C, and release of uracil was determined by precipitation with trichloroacetic acid.

View larger version (9K): [in this window] [in a new window]   Fig. 3.   Lineweaver-Burk plot for the determination of Km for the release of uracil from a double-stranded DNA substrate containing 3H-labeled uracil. Substrate range, 0.03 to 0.6 µM; Km = 0.5 µM.

Activity on Single-stranded DNA-- The activity of the expressed protein was also measured in a single-stranded DNA substrate containing ³H-labeled uracil substituted for thymine and was measured at 95 °C. The K_m for release of uracil from this substrate was also determined from Lineweaver-Burk analysis to be 0.5 µM, over a substrate range of 0.03 to 0.6 µM. The kinetic constants (K_m, k_cat, and k_cat/K_m determined for both double- and single-stranded DNA) are shown in Table I.

Substrate Specificity of AFUDG-- To determine if AFUDG could remove uracil opposite guanine, as would occur in DNA following cytosine deamination, double-stranded oligonucleotide substrates containing either a single U/A or U/G base pair were prepared, and the activity of AFUDG on these substrates was determined. These substrates are subject to alkaline cleavage at the internal AP site following removal of uracil (16, 19, 20). The substrates were treated at 50 °C with AFUDG to prevent thermal melting of the duplex oligonucleotides. As seen in Fig. 4, the enzyme was capable of removing uracil from both types of substrates, as seen by the formation of an 11-mer with an unsaturated sugar-phosphate group at the 3' end (21) when the reaction products are resolved on a denaturing gel. The enzyme did not remove thymine from an analogous oligonucleotide substrate containing a T/G base pair under identical reaction conditions. These results suggest that AFUDG has similar enzymatic functions as the T. maritima UDG (14).

View larger version (66K): [in this window] [in a new window]   Fig. 4.   AFUDG removes uracil from double-stranded oligonucleotides containing either a U/G or U/A base pair. The 30-mer double-stranded oligonucleotides (20 fmol each) were incubated in a 20-µl reaction mixture containing 50 mM MOPS-KOH, pH 7.8, 0.1 mM EDTA, 1 mM DTT, 100 µg/ml BSA, 10 ng of AFUDG, for 10 min at 50 °C. The reactions were stopped by the addition 20 µl of 0.1 M NaOH, and the samples were heated at 90 °C for 30 min to cleave the phosphodiester bonds at the abasic sites. The samples were resolved on a 20% polyacrylamide gel containing 7 M urea. Lane 1, (U/A) 30-mer not treated with enzyme; lane 2, (U/A) 30-mer incubated with enzyme; lane 3, (T/G) 30-mer not treated with enzyme; lane 4, (T/G) 30-mer incubated with enzyme; lane 5, (U/G) 30-mer not treated with enzyme; lane 6, (U/G) 30-mer incubated with enzyme.

Product Inhibition of AFUDG-- It has been shown previously that uracil-DNA glycosylases are product-inhibited by uracil and, in most cases, AP sites present in DNA (22-24). As seen in Fig. 5, an increasing concentration of uracil up to 10 mM resulted in up to a 40% reduction in the removal of uracil. In contrast, 2-deoxyribose 5-phosphate (dRp) at a concentration of 5 mM resulted in less than a 10% reduction of activity. To determine if AP sites present in DNA were inhibitory, 30-mer oligonucleotides as described above containing AP sites (either opposite G or A) were prepared and were incubated with AFUDG and the double-stranded DNA substrate containing ³H-labeled uracil. As shown in Fig. 6, oligonucleotides containing AP sites opposite both A or G were inhibitory (greater than 50% inhibition with a concentration of 4 µM and higher).

View larger version (7K): [in this window] [in a new window]   Fig. 5.   Uracil inhibits the activity of AFUDG. The release of uracil from a double-stranded DNA substrate containing 3H-labeled uracil was determined in a 10-min reaction at 70 °C in the presence of uracil base. The release of uracil was determined by precipitation with trichloroacetic acid.

View larger version (8K): [in this window] [in a new window]   Fig. 6.   AP sites inhibit the activity of AFUDG. The release of uracil from a double-stranded DNA substrate containing 3H-labeled uracil was determined in a 10-min reaction at 50 °C in the presence of AP site-containing oligonucleotides. The release of uracil was determined by precipitation with trichloroacetic acid. , 30-mer containing AP site opposite A; , 30-mer containing AP site opposite G.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

We have described a novel uracil-DNA glycosylase found in A. fulgidus that functions similarly to the E. coli UDG and the T. maritima UDG but with an extremely high degree of heat stability. The enzyme is a member of a new class of UDGs that have functional similarity to the E. coli/eukaryotic/DNA-containing virus class of enzymes but differ at the primary amino acid sequence level. This class of enzymes has been found in both archaea as well as eubacteria and in both thermophiles and mesophiles (14).

The A. fulgidus UDG is the first enzyme of its type to be identified and characterized from archaea. Fig. 7 shows an alignment of multiple amino acid sequences identified for putative homologues of AFUDG in archaeal species. In addition to A. fulgidus, homologues have been identified so far in Pyrococcus horikoshii, Pyrococcus abyssi, and Aeropyrum pernix.

View larger version (77K): [in this window] [in a new window]   Fig. 7.   Amino acid alignment of A. fulgidus uracil-DNA glycosylase with putative homologues from P. horikoshii, P., and A. pernix. Homologous ORFs were identified by using TBLASTN software at the National Center for Biotechnology Information (17). The amino acid sequences were aligned using the program CLUSTALW (25). Black boxes indicate identity and shaded boxes indicate conservative changes.

The gene encoding AFUDG was identified initially as a homologue of a DNA polymerase from the bacteriophage SP01 that infects B. subtilis (15, 18). This phage substitutes hydroxymethyluracil for thymine in its DNA (26, 27). AFUDG demonstrated no DNA polymerase activity and is considerably smaller in size (21 versus 106 kDa) than the SP01 DNA polymerase; however, it shows considerable homology to the amino-terminal end of the SP01 DNA polymerase. Whether AFUDG is capable of removing hydroxymethyluracil from DNA remains to be investigated.

AFUDG was found to be inhibited by both uracil as well as AP sites present in DNA. The degree of inhibition by an AP site was essentially the same if the AP site was opposite A or opposite G. Inclusion of sugar-phosphate (dRp) in the reaction did not effectively inhibit the activity of AFUDG, suggesting the enzyme requires an intact AP site for recognition. Other UDG activities are also inhibited by intact AP sites; however, it has been demonstrated that a form of the human mitochondrial enzyme exists that is resistant to AP site inhibition (24).

We believe that AFUDG is used in the first step for the removal of uracil in a base excision repair pathway in A. fulgidus and suggests a conservation of the UDG-initiated base excision repair pathway in archaea. Recently, it has been demonstrated that archaeal DNA polymerases can recognize uracil residues in the template strand and stall DNA synthesis (28). It is possible that archaeal DNA polymerases may interact directly with the uracil-DNA glycosylase, thus providing a role for this enzyme in removing uracil residues that may result at replication forks.

	FOOTNOTES

^* This work was supported by NCI Grant CA52025 from the National Institutes of Health.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Depts. of Radiology and Radiation Oncology, Albert Einstein College of Medicine, 1300 Morris Park Ave., Bronx, NY 10461. Tel.: 718-430-2239; Fax: 718-430- 4039; E-mail: frankin@aecom.yu.edu.

Published, JBC Papers in Press, April 20, 2000, DOI 10.1074/jbc.M001995200

	ABBREVIATIONS

The abbreviations used are: UDG, uracil-DNA glycosylase; AP, apurinic/apyrimidinic; AFUDG, A. fulgidus uracil-DNA glycosylase; dRp, deoxyribose phosphate; DTT, dithiothreitol; BSA, bovine serum albumin; PCR, polymerase chain reaction; ORF, open reading frame; MOPS, 4-morpholinepropanesulfonic acid.

	REFERENCES

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