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• W2024758879 abstract "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. 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. uracil-DNA glycosylase apurinic/apyrimidinic A. fulgidusuracil-DNA glycosylase deoxyribose phosphate dithiothreitol bovine serum albumin polymerase chain reaction open reading frame 4-morpholinepropanesulfonic acid Uracil-DNA glycosylase (UDG)1 is a ubiquitous enzyme found in most eukaryotes and prokaryotes (1.Mosbaugh D.W. Bennett S.E. Prog. Nucleic Acids Res. Mol. Biol. 1994; 48: 315-370Crossref PubMed Scopus (97) Google Scholar, 2.Krokan H.E. Standal R. Slupphaug G. Biochem. J. 1997; 325: 1-16Crossref PubMed Scopus (726) Google Scholar, 3.Cunningham R.P. Mutat. Res. 1997; 383: 189-196Crossref PubMed Scopus (96) Google Scholar). This enzyme removes uracil that is present in DNA either due to deamination of cytosine or misincorporation of dUMP in place of dTMP (4.Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4336) Google Scholar, 5.Tye B.K. Nyman P.O. Lehman I.R. Hochhauser S. Weiss B. Proc. Natl. Acad. Sci. U. S. A. 1977; 74: 154-157Crossref PubMed Scopus (224) Google Scholar) 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 bothEscherichia coli and from eukaryotic cells; the crystal structures of the E. coli, human, and herpes simplex virus UDGs have been solved (6.Mol C.D. Arvai A.S. Slupphaug G. Kovil B. Alseth I. Krokan H.E. Tainer J.A. Cell. 1995; 80: 869-878Abstract Full Text PDF PubMed Scopus (340) Google Scholar, 7.Savva R. McAuley-Hecht K. Brown T. Pearl L. Nature. 1995; 373: 487-493Crossref PubMed Scopus (387) Google Scholar, 8.Ravishankar R. Sagar M.B Roy S. Purnapatre K. Hanada P. Varshney U. Vijayan M. Nucleic Acids Res. 1998; 26: 4880-4887Crossref PubMed Scopus (54) Google Scholar). 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.Olsen L.C. Aasland R. Wittwer C.U. Krokan H.E. Helland D.E. EMBO J. 1989; 8: 3121-3125Crossref PubMed Scopus (169) Google Scholar). UDG activities have been shown to be present in several thermophiles (10.Kaboev O.K. Luchkina L.A. Akhmedov A.T. Bekker M.L. FEBS Lett. 1981; 132: 337-340Crossref PubMed Scopus (10) Google Scholar, 11.Warner H, R. J. Bacteriol. 1983; 154: 1451-1454Crossref PubMed Google Scholar, 12.Koulis A. Cowan D.A. Pearl L.H. Savva R. FEMS Microbiol. Lett. 1996; 143: 267-271Crossref PubMed Google Scholar). However, several bacterial genomes lack sequences complementary to the E. coli ung gene (13.Aravind L. Walker D.R. Koonin E.V. Nucleic Acids Res. 1999; 27: 1223-1242Crossref PubMed Scopus (484) Google Scholar). 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.Sandigursky M. Franklin W.A. Curr. Biol. 1999; 9: 531-534Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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.Klenk H.-P. Clayton R.A. Tomb J.-F. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. et al.Nature. 1997; 390: 364-370Crossref PubMed Scopus (1203) Google Scholar). 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. BW310 (l-, ung-1, relA1, spoT1, thi-, obtained from E. coli Genetic Stock Center, Yale University) was lysogenized with λDE3 using the λ lysogenation kit from Novagen. The plasmid pET28a was obtained from Novagen. PCR was carried out using a pUC18 plasmid containing an insert of A. fulgidusgenomic 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 andBamHI 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). 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 theA 600 of the culture reached 0.8. Isopropyl-1-thio-β-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 containing 3H-labeled uracil was prepared by nick translation of calf thymus DNA as described previously (14.Sandigursky M. Franklin W.A. Curr. Biol. 1999; 9: 531-534Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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.Sandigursky M. Franklin W.A. Curr. Biol. 1999; 9: 531-534Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar, 16.Varshney U. van de Sande J.H. Biochemistry. 1991; 30: 4055-4061Crossref PubMed Scopus (97) Google Scholar). 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 (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. 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. An open reading frame (ORF) analogous to the UDG gene from T. maritima (14.Sandigursky M. Franklin W.A. Curr. Biol. 1999; 9: 531-534Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar) was identified following a BLAST (17.Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Miller W. Lippman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59879) Google Scholar) search of the A. fulgidusgenomic DNA (15.Klenk H.-P. Clayton R.A. Tomb J.-F. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. et al.Nature. 1997; 390: 364-370Crossref PubMed Scopus (1203) Google Scholar). 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.Scarlato V. Gargano S. Gene (Amst.). 1992; 118: 109-113Crossref PubMed Scopus (15) Google Scholar). 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. 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.Figure 3Lineweaver-Burk plot for the determination ofK m for the release of uracil from a double-stranded DNA substrate containing 3H-labeled uracil. Substrate range, 0.03 to 0.6 μm;K m = 0.5 μm.View Large Image Figure ViewerDownload Hi-res image Download (PPT) The activity of the expressed protein was also measured in a single-stranded DNA substrate containing3H-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, andk cat/K m determined for both double- and single-stranded DNA) are shown in TableI.Table IKinetic constants for AFUDGK mk catk cat/K mμmmin −1μm −1 min −1dsDNA0.555110ssDNA0.53363Kinetic parameters were determined from direct linear plots (Lineweaver-Burk). k cat was calculated fromV max using a molecular mass of 22,718 daltons for AFUDG. Open table in a new tab Kinetic parameters were determined from direct linear plots (Lineweaver-Burk). k cat was calculated fromV max using a molecular mass of 22,718 daltons for 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.Varshney U. van de Sande J.H. Biochemistry. 1991; 30: 4055-4061Crossref PubMed Scopus (97) Google Scholar, 19.Eftedal I. Guddal P.H. Slupphaug G. Volden G. Krokan H.E. Nucleic Acids Res. 1993; 21: 2095-2101Crossref PubMed Scopus (68) Google Scholar, 20.Dianov G. Price A. Lindahl T. Mol. Cell. Biol. 1992; 12: 1605-1612Crossref PubMed Scopus (262) Google Scholar). 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.Mazumder A. Gerlt J.A. Absalon M.J. Stubbe J. Cunningham R.P. Withka J. Biochemistry. 1991; 30: 1119-1126Crossref PubMed Scopus (172) Google Scholar) 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.Sandigursky M. Franklin W.A. Curr. Biol. 1999; 9: 531-534Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). It has been shown previously that uracil-DNA glycosylases are product-inhibited by uracil and, in most cases, AP sites present in DNA (22.Lindahl T. Ljungquist S. Siegert W. Nyberg B. Sperens B. J. Biol. Chem. 1977; 252: 3286-3294Abstract Full Text PDF PubMed Google Scholar, 23.Domena J.D. Timmer R.T. Dicharry S.A. Mosbaugh D.W. Biochemistry. 1988; 27: 6742-6751Crossref PubMed Scopus (69) Google Scholar, 24.Bharati S. Korokan H.E. Kristiansen L. Otterlei M. Slupphaug G. Nucleic Acids Res. 1998; 26 (3959): 4953Crossref PubMed Scopus (50) Google Scholar). 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 containing3H-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).Figure 6AP sites inhibit the activity of AFUDG. The release of uracil from a double-stranded DNA substrate containing3H-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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.Sandigursky M. Franklin W.A. Curr. Biol. 1999; 9: 531-534Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). 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. The gene encoding AFUDG was identified initially as a homologue of a DNA polymerase from the bacteriophage SP01 that infects B. subtilis (15.Klenk H.-P. Clayton R.A. Tomb J.-F. White O. Nelson K.E. Ketchum K.A. Dodson R.J. Gwinn M. Hickey E.K. Peterson J.D. et al.Nature. 1997; 390: 364-370Crossref PubMed Scopus (1203) Google Scholar, 18.Scarlato V. Gargano S. Gene (Amst.). 1992; 118: 109-113Crossref PubMed Scopus (15) Google Scholar). This phage substitutes hydroxymethyluracil for thymine in its DNA (26.Glassberg J. Slomiany R.A. Stewart C.R. J. Virol. 1977; 21: 54-60Crossref PubMed Google Scholar, 27.Glasberg J. Franck M. Stewart C.R. J. Virol. 1977; 21: 147-152Crossref PubMed Google Scholar). 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.Bharati S. Korokan H.E. Kristiansen L. Otterlei M. Slupphaug G. Nucleic Acids Res. 1998; 26 (3959): 4953Crossref PubMed Scopus (50) Google Scholar). 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.Greagg M.A. Fogg M.J. Panayotou G. Evans S.J. Connolly B.A. Pearl L.H. Proc. Natl. Acad. Sci. U. S. A. 1999; 16: 9045-9050Crossref Scopus (106) Google Scholar). 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." @default.
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• W2024758879 title "Uracil-DNA Glycosylase in the Extreme Thermophile Archaeoglobus fulgidus" @default.
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