Uracil-DNA Glycosylase in the Extreme Thermophile Archaeoglobus fulgidus

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 E. coli , eukaryotes, and DNA-containing viruses. The A. fulgidus UDG is extremely thermostable, maintaining full activity after heating for 1.5 hours at 95 o 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 (AP) 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. 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 Thermotoga maritima , yet is considerably more heat stable. These findings suggest a conserved mechanism of uracil base excision repair in archaea.


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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 Thermotoga maritima, yet is considerably more heat stable. These findings suggest a conserved mechanism of uracil base excision repair in archaea.

Cloning of the Archaeoglobus 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 Bam HI 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 34mg/ml kanamycin (LB-kan) and was grown overnight at 37 o C. The overnight culture was diluted 1:50 with fresh LB-kan medium and was grown at 37 o C until the A 600 of the culture reached 0.8. IPTG was then added to a final concentration of 1 mM, and the culture was incubated for an additional 3 hr at 30 o C. Cells were pelleted by centrifugation at 3,000 x g for 5 min at 4 o C and then resuspended in 2 ml ice-cold buffer containing 5 mM imidazole, 500 mM NaCl and 20 mM Tris-HCl, pH 7.9 (1x binding buffer). Cells were lysed by sonication with 4 x 10 second bursts. The sonicate was clarified by centrifugation at 12,000 x g at 4 o 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 Ni 2+ column (Novagen), which was subsequently washed with 12 ml 1x binding buffer. Protein was eluted from the column with buffer containing 60 mM (6 ml), 100 mM, 250 mM 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 (Pharmacia), and eluted with 3.5 ml 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 (Pharmacia) 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. Reactions were stopped by the addition of 110 µl 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.
Activity on single-stranded DNA--The activity of the expressed protein was also measured in a single-stranded DNA substrate containing [ 3 H]-labeled uracil substituted for thymine and was measured at 95 o 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 µM 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, doublestranded 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 o 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).
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)(23)(24). As seen in Fig. 5, an increasing concentration of uracil up to 10 mM Whether AFUDG is capable of removing hydroyxmethyluracil from DNA remains to be investigated.

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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 UDGinitiated 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.