In vivo repair of alkylating and oxidative DNA damage in the mitochondrial and nuclear genomes of wild-type and glycosylase-deficient Caenorhabditis elegans

Base excision repair (BER) is an evolutionarily conserved DNA repair pathway that is critical for repair of many of the most common types of DNA damage generated both by endogenous metabolic pathways and exposure to exogenous stressors such as pollutants. Caenorhabditis elegans is an increasingly important model organism for the study of DNA damage-related processes including DNA repair, genotoxicity, and apoptosis, but BER is not well understood in this organism, and has not previously been measured in vivo. We report robust BER in the nuclear genome and slightly slower damage removal from the mitochondrial genome; in both cases the removal rates are comparable to those observed in mammals. However we could detect no deficiency in BER in the nth-1 strain, which carries a deletion in the only glycosylase yet described in C. elegans that repairs oxidative DNA damage. We also failed to detect increased lethality or growth inhibition in nth-1 nematodes after exposure to oxidative or alkylating damage, suggesting the existence of at least one additional as-yet undetected glycosylase.     

The mouse uracil-DNA glycosylase gene: isolation of cDNA and genomic clones and mapping ung to mouse chromosome 5

Uracil-DNA glycosylase (UDG) is the enzyme responsible for the first step in the base-excision repair pathway that specifically removes uracil from DNA. Here we report the isolation of the cDNA and genomic clones for the mouse uracil-DNA glycosylase gene (ung) homologous to the major placental uracil-DNA glycosylase gene (UNG) of humans. The complete characterization of the genomic organization of the mouse uracil-DNA glycosylase gene shows that the entire mRNA coding region for the 1.83-kb cDNA of the mouse ung gene is contained in an 8.2-kb SstI genomic fragment which includes six exons and five introns. The cDNA encodes a predicted uracil-DNA glycosylase (UDG) protein of 295 amino acids (33 kDa) that is highly similar to a group of UDGs that have been isolated from a wide variety of organisms. The mouse ung gene has been mapped to mouse chromosome 5 using fluorescence in situ hybridization (FISH).     

Base excision repair initiation revealed by crystal structures and binding kinetics of human uracil-DNA glycosylase with DNA.

Three high-resolution crystal structures of DNA complexes with wild-type and mutant human uracil-DNA glycosylase (UDG), coupled kinetic characterizations and comparisons with the refined unbound UDG structure help resolve fundamental issues in the initiation of DNA base excision repair (BER): damage detection, nucleotide flipping versus extrahelical nucleotide capture, avoidance of apurinic/apyrimidinic (AP) site toxicity and coupling of damage-specific and damage-general BER steps. Structural and kinetic results suggest that UDG binds, kinks and compresses the DNA backbone with a 'Ser-Pro pinch' and scans the minor groove for damage. Concerted shifts in UDG simultaneously form the catalytically competent active site and induce further compression and kinking of the double-stranded DNA backbone only at uracil and AP sites, where these nucleotides can flip at the phosphate-sugar junction into a complementary specificity pocket. Unexpectedly, UDG binds to AP sites more tightly and more rapidly than to uracil-containing DNA, and thus may protect cells sterically from AP site toxicity. Furthermore, AP-endonuclease, which catalyzes the first damage-general step of BER, enhances UDG activity, most likely by inducing UDG release via shared minor groove contacts and flipped AP site binding. Thus, AP site binding may couple damage-specific and damage-general steps of BER without requiring direct protein-protein interactions.     

Affinity Purification and Comparative Analysis of Two Distinct Human Uracil-DNA Glycosylases

Evidence is presented on two forms of uracil-DNA glycosylase (UDG1 and UDG2) that exist in human cells. We have developed an affinity technique to isolate uracil-DNA glycosylases from HeLa cells. This technique relies on the use of a uracil-DNA glycosylase inhibitor (Ugi) produced by theBacillus subtilisbacteriophage, PBS2. Affinity-purified preparations of uracil-DNA glycosylase, derived from total HeLa cell extracts, reveal a group of bands in the 36,000 molecular weight range and a single 30,000 molecular weight band when analyzed by SDS–PAGE and silver staining. In contrast, only the 30,000 molecular weight band is seen in HeLa mitochondrial preparations. Separation of HeLa cell nuclei from the postnuclear supernatant reveals that uracil-DNA glycosylase activity is evenly distributed between the nuclear compartment and the postnuclear components of the cell. Immunostaining of a nuclear extract with antisera to UDG1 indicates that the nuclear associated uracil-DNA glycosylase activity is not associated with the highly conserved uracil-DNA glycosylase, UDG1. With the use of Ugi-Sepharose affinity chromatography, we show that a second and distinct uracil-DNA glycosylase is associated with the nuclear compartment. Immunoblot analysis, utilizing antisera generated against UDG1, reveals that the 30,000 molecular weight protein and a protein in the 36,000 range share common epitopes. Cycloheximide treatment of HeLa cells indicates that upon inhibition of protein synthesis, the higher molecular weight species disappears and is apparently posttranslationally processed into a lower molecular weight form. This is substantiated by mitochondrial import studies which reveal thatin vitroexpressed UDG1 becomes resistant to trypsin treatment within 15 min of incubation with mitochondria. Within this time frame, a lower molecular weight form of uracil-DNA glycosylase appears and is associated with the mitochondria. Antibodies generated against peptides from specific regions of the cyclin-like uracil-DNA glycosylase (UDG2), demonstrate that this nuclear glycosylase is a phosphoprotein with a molecular weight in the range of 36,000. SDS–PAGE analysis of Ugi affinity-purified and immunoprecipitated UDG2 reveals two closely migrating phosphate-containing species, indicating that UDG2 either contains multiple phosphorylation sites (resulting in heterogeneous migration) or that two distinct forms of UDG2 exist in the cell. Cell staining of various cultured human cell lines corroborates the finding that UDG1 is largely excluded from the nucleus and that UDG2 resides mainly in the nucleus. Our results indicate that UDG1 is targeted to the mitochondria and undergoes proteolytic processing typical of resident mitochondrial proteins that are encoded by nuclear DNA. These results also indicate that the cyclin-like uracil-DNA glycosylase (UDG2) may be a likely candidate for the nuclear located base-excision repair enzyme.     

New family of deamination repair enzymes in uracil-DNA glycosylase superfamily

DNA glycosylases play a major role in the repair of deaminated DNA damage. Previous investigations identified five families within the uracil-DNA glycosylase (UDG) superfamily. All enzymes within the superfamily studied thus far exhibit uracil-DNA glycosylase activity. Here we identify a new class of DNA glycosylases in the UDG superfamily that lacks UDG activity. Instead, these enzymes act as hypoxanthine-DNA glycosylases in vitro and in vivo. Molecular modeling and structure-guided mutational analysis allowed us to identify a unique catalytic center in this class of DNA glycosylases. Based on unprecedented biochemical properties and phylogenetic analysis, we propose this new class of DNA repair glycosylases that exists in bacteria, archaea, and eukaryotes as family 6 and designate it as the hypoxanthine-DNA glycosylase family. This study demonstrates the structural evolvability that underlies substrate specificity and catalytic flexibility in the evolution of enzymatic function.     

Uracil-DNA glycosylase causes 5-bromodeoxyuridine photosensitization in Escherichia coli K-12.

An Escherichia coli uracil-DNA glycosylase-defective mutant (ung-1 thyA) was more resistant than its wild-type counterpart (ung+ thyA) to the killing effect of UV light when cultured in medium containing 5-bromouracil or 5-bromo-2'-deoxyuridine (BrdUrd). The phenotype of resistance to BrdUrd photosensitization and the uracil-DNA glycosylase deficiency appeared to be 100% cotransduced by P1 phage. During growth with BrdUrd, both strains exhibited similar growth rates and 5-bromouracil incorporation into DNA. The resistant phenotype of the ung-1 mutant was observed primarily during the stationary phase. In cells carrying 5-bromouracil-substituted DNA, mutations causing resistance to rifampin and valine were induced by UV irradiation at a higher frequency in the wild type than in the ung-1 mutant. This Ung-dependent UV mutagenesis required UmuC function. These results suggest that the action of the uracil-DNA glycosylase on UV-irradiated 5-bromouracil-substituted DNA produces lethal and mutagenic lesions. The BrdUrd photosensitization-resistant phenotype allowed us to develop a new, efficient method for enriching and screening ung mutants.     

Plant mitochondria possess a short-patch base excision DNA repair pathway

Despite constant threat of oxidative damage, sequence drift in mitochondrial and chloroplast DNA usually remains very low in plant species, indicating efficient defense and repair. Whereas the antioxidative defense in the different subcellular compartments is known, the information on DNA repair in plant organelles is still scarce. Focusing on the occurrence of uracil in the DNA, the present work demonstrates that plant mitochondria possess a base excision repair (BER) pathway. In vitro and in organello incision assays of double-stranded oligodeoxyribonucleotides showed that mitochondria isolated from plant cells contain DNA glycosylase activity specific for uracil cleavage. A major proportion of the uracil–DNA glycosylase (UDG) was associated with the membranes, in agreement with the current hypothesis that the DNA is replicated, proofread and repaired in inner membrane-bound nucleoids. Full repair, from uracil excision to thymidine insertion and religation, was obtained in organello following import of a uracil-containing DNA fragment into isolated plant mitochondria. Repair occurred through single nucleotide insertion, which points to short-patch BER. In vivo targeting and in vitro import of GFP fusions showed that the putative UDG encoded by the At3g18 630 locus might be the first enzyme of this mitochondrial pathway in Arabidopsis thaliana.     

Staphylococcus aureus protein SAUGI acts as a uracil-DNA glycosylase inhibitor

DNA mimic proteins are unique factors that control the DNA binding activity of target proteins by directly occupying their DNA binding sites. The extremely divergent amino acid sequences of the DNA mimics make these proteins hard to predict, and although they are likely to be ubiquitous, to date, only a few have been reported and functionally analyzed. Here we used a bioinformatic approach to look for potential DNA mimic proteins among previously reported protein structures. From ∼14 candidates, we selected the Staphylococcus conserved hypothetical protein SSP0047, and used proteomic and structural approaches to show that it is a novel DNA mimic protein. In Staphylococcus aureus, we found that this protein acts as a uracil-DNA glycosylase inhibitor, and therefore named it S. aureus uracil-DNA glycosylase inhibitor (SAUGI). We also determined and analyzed the complex structure of SAUGI and S. aureus uracil-DNA glycosylase (SAUDG). Subsequent BIAcore studies further showed that SAUGI has a high binding affinity to both S. aureus and human UDG. The two uracil-DNA glycosylase inhibitors (UGI and p56) previously known to science were both found in Bacillus phages, and this is the first report of a bacterial DNA mimic that may regulate SAUDG’s functional roles in DNA repair and host defense.     

Mitochondrial base excision repair of uracil and AP sites takes place by single-nucleotide insertion and long-patch DNA synthesis

Base excision repair (BER) corrects a variety of small base lesions in DNA. The UNG gene encodes both the nuclear (UNG2) and the mitochondrial (UNG1) forms of the human uracil-DNA glycosylase (UDG). We prepared mitochondrial extracts free of nuclear BER proteins from human cell lines. Using these extracts we show that UNG is the only detectable UDG in mitochondria, and mitochondrial BER (mtBER) of uracil and AP sites occur by both single-nucleotide insertion and long-patch repair DNA synthesis. Importantly, extracts of mitochondria carry out repair of modified AP sites which in nuclei occurs through long-patch BER. Such lesions may be rather prevalent in mitochondrial DNA because of its proximity to the electron transport chain, the primary site of production of reactive oxygen species. Furthermore, mitochondrial extracts remove 5' protruding flaps from DNA which can be formed during long-patch BER, by a "flap endonuclease like" activity, although flap endonuclease (FEN1) is not present in mitochondria. In conclusion, combined short- and long-patch BER activities enable mitochondria to repair a broader range of lesions in mtDNA than previously known.     

Arabidopsis Uracil DNA Glycosylase (UNG) Is Required for Base Excision Repair of Uracil and Increases Plant Sensitivity to 5-Fluorouracil

Uracil in DNA arises by misincorporation of dUMP during replication and by hydrolytic deamination of cytosine. This common lesion is actively removed through a base excision repair (BER) pathway initiated by a uracil DNA glycosylase (UDG) activity that excises the damage as a free base. UDGs are classified into different families differentially distributed across eubacteria, archaea, yeast, and animals, but remain to be unambiguously identified in plants. We report here the molecular characterization of AtUNG (Arabidopsis thaliana uracil DNA glycosylase), a plant member of the Family-1 of UDGs typified by Escherichia coli Ung. AtUNG exhibits the narrow substrate specificity and single-stranded DNA preference that are characteristic of Ung homologues. Cell extracts from atung^−/− mutants are devoid of UDG activity, and lack the capacity to initiate BER on uracil residues. AtUNG-deficient plants do not display any apparent phenotype, but show increased resistance to 5-fluorouracil (5-FU), a cytostatic drug that favors dUMP misincorporation into DNA. The resistance of atung^−/− mutants to 5-FU is accompanied by the accumulation of uracil residues in DNA. These results suggest that AtUNG excises uracil in vivo but generates toxic AP sites when processing abundant U:A pairs in dTTP-depleted cells. Altogether, our findings point to AtUNG as the major UDG activity in Arabidopsis.     

W-mutagenesis in the bisulfite-treated lambda phage

Survival of phage lambda cI857 inactivated by bisulfite (pH 5.6, 37 degrees C) is higher (the dose modification factor approx. 1.2) and frequency of bisulfite-induced c-mutations 2-4-fold lower on the lawn of the wild-type strain ung+, as compared to ung-1 mutant deficient in uracil-DNA glycosylase. Irradiation of host cells by a moderate UV dose inducing SOS repair system enhances the frequency of bisulfite-induced c-mutations 2-3-fold in the wild-type (ung+) host, but not in the ung-1 mutant. It is suggested that W-mutagenesis in bisulfite-treated lambda phage in the ung+ cells is due to SOS repair of apyrimidinic sites which are produced during excision of uracil residues, the products of cytosine deamination.     

Trypanosoma cruzi contains a single detectable uracil-DNA glycosylase and repairs uracil exclusively via short patch base excision repair

Enzymes involved in genomic maintenance of human parasites are attractive targets for parasite-specific drugs. The parasitic protozoan Trypanosoma cruzi contains at least two enzymes involved in the protection against potentially mutagenic uracil, a deoxyuridine triphosphate nucleotidohydrolase (dUTPase) and a uracil-DNA glycosylase belonging to the highly conserved UNG-family. Uracil-DNA glycosylase activities excise uracil from DNA and initiate a multistep base-excision repair (BER) pathway to restore the correct nucleotide sequence. Here we report the biochemical characterisation of T.cruzi UNG (TcUNG) and its contribution to the total uracil repair activity in T.cruzi. TcUNG is shown to be the major uracil-DNA glycosylase in T.cruzi. The purified recombinant TcUNG exhibits substrate preference for removal of uracil in the order ssU>U:G>U:A, and has no associated thymine-DNA glycosylase activity. T.cruzi apparently repairs U:G DNA substrate exclusively via short-patch BER, but the DNA polymerase involved surprisingly displays a vertebrate POLdelta-like pattern of inhibition. Back-up UDG activities such as SMUG, TDG and MBD4 were not found, underlying the importance of the TcUNG enzyme in protection against uracil in DNA and as a potential target for drug therapy.     

The Hyperthermophilic Euryarchaeon Archaeoglobus fulgidus Repairs Uracil by Single-Nucleotide Replacement

Hydrolytic deamination of cytosine to uracil in cellular DNA is a major source of C-to-T transition mutations if uracil is not repaired by the DNA base excision repair (BER) pathway. Since deamination increases rapidly with temperature, hyperthermophiles, in particular, are expected to succumb to such damage. There has been only one report of crenarchaeotic BER showing strong similarities to that in most eukaryotes and bacteria for hyperthermophilic Archaea. Here we report a different type of BER performed by extract prepared from cells of the euryarchaeon Archaeoglobus fulgidus. Although immunodepletion showed that the monofunctional family 4 type of uracil-DNA glycosylase (UDG) is the principal and probably only UDG in this organism, a β-elimination mechanism rather than a hydrolytic mechanism is employed for incision of the abasic site following uracil removal. The resulting 3′ remnant is removed by efficient 3′-phosphodiesterase activity followed by single-nucleotide insertion and ligation. The finding that repair product formation is stimulated similarly by ATP and ADP in vitro raises the question of whether ADP is more important in vivo because of its higher heat stability.After depurination, hydrolytic deamination of cytosine to uracil is the most frequent event that damages DNA (36), and it results in G·C-to-A·T transition mutations if the damage is not repaired. In addition, some dUTP molecules escape hydrolysis by dUTPase, which results in a certain amount of dUMP introduced into DNA opposite adenine during replication (32). Irrespective of the mode of appearance, all cells contain uracil-DNA glycosylase (UDG) (EC 3.2.2.3) enzymes to remove uracil from DNA (17). The resulting abasic or apurinic/apyrimidinic (AP) site can subsequently be removed, and the integrity of the DNA can be restored by the so-called base excision repair (BER) pathway, which consists in its simplest form of the sequential actions of 5′-acting AP endonuclease, 5′-deoxyribose phosphate (dRP) lyase, DNA polymerase, and DNA ligase. The BER pathway can be initiated by one of several DNA glycosylases with different substrate specificities (17, 36, 57), and quantitatively it is the most important repair mechanism for the removal of spontaneously generated base modifications. Genes encoding bacterial and eukaryotic UDGs exhibiting significant selectivity for uracil have been cloned and sequenced in the last 2 decades, and the results have demonstrated that there is a high degree of conservation between distantly related species. Family 1 UDGs (for a review of UDG families 1 to 3, see reference 44), typified by the Escherichia coli Ung enzyme (37), recognize uracil in an extrahelical or flipped-out conformation in double-stranded DNA (dsDNA) and single-stranded DNA (ssDNA). Several family 1 enzymes have been extensively characterized, both structurally and at the cell and organism levels. Family 2 UDGs, which includes E. coli Mug and mammalian thymine-DNA glycosylase, are mismatch specific and recognize guanine on the complementary strand rather than the lesion itself and thus are inactive with ssDNA. Family 3 UDGs, typified by the SMUG1 enzyme of human cells, have similar substrate requirements but exhibit a stronger preference for uracil in ssDNA than family 1 enzymes (17, 27, 57, 67).UDG activity in hyperthermophilic microorganisms was first reported in 1996 (33). Three years later, Sandigursky and Franklin (47) cloned and overexpressed an open reading frame (ORF) of the hyperthermophilic bacterium Thermotoga maritima that typifies the family 4 UDGs that are able to remove uracil from U·G and U·A base pairs, as well as from ssDNA. By means of homology searches, these workers found ORFs homologous to the T. maritima UDG gene in several prokaryotic genomes, including that of the hyperthermophilic archaeon Archaeoglobus fulgidus, a strict anaerobe that grows optimally at 83°C (60; for a review of DNA repair in hyperthermophilic archaea, see reference 20). Subsequently, they cloned and overexpressed the A. fulgidus ORF in E. coli by producing a His-tagged fusion protein. As expected, the purified A. fulgidus recombinant Afung (rAfung) protein exhibited UDG activity (48). However, whether Afung is the major UDG of A. fulgidus or is just a minor glycosylase with uracil-releasing ability remained to be determined.As a continuation of previous biochemical and physicochemical studies (31) of non-His-tagged rAfung protein, here we characterized a family 4 UDG in archaeon cell extract. The abundance of Afung in vivo was determined, and evidence indicates that this enzyme is the principal UDG of A. fulgidus. Here we also describe the mechanism of dUMP repair employed by this euryarchaeon, which differs in important ways from the mechanism reported for the crenarchaeon Pyrobaculum aerophilum (50).     

Protein p56 from the Bacillus subtilis phage phi29 inhibits DNA-binding ability of uracil-DNA glycosylase

Protein p56 (56 amino acids) from the Bacillus subtilis phage ϕ29 inactivates the host uracil-DNA glycosylase (UDG), an enzyme involved in the base excision repair pathway. At present, p56 is the only known example of a UDG inhibitor encoded by a non-uracil containing viral DNA. Using analytical ultracentrifugation methods, we found that protein p56 formed dimers at physiological concentrations. In addition, circular dichroism spectroscopic analyses revealed that protein p56 had a high content of β-strands (around 40%). To understand the mechanism underlying UDG inhibition by p56, we carried out in vitro experiments using the Escherichia coli UDG enzyme. The highly acidic protein p56 was able to compete with DNA for binding to UDG. Moreover, the interaction between p56 and UDG blocked DNA binding by UDG. We also demonstrated that Ugi, a protein that interacts with the DNA-binding domain of UDG, was able to replace protein p56 previously bound to the UDG enzyme. These results suggest that protein p56 could be a novel naturally occurring DNA mimicry.     

Identification of a prototypical single-stranded uracil DNA glycosylase from Listeria innocua

A recent phylogenetic study on UDG superfamily estimated a new clade of family 3 enzymes (SMUG1-like), which shares a lower homology with canonic SMUG1 enzymes. The enzymatic properties of the newly found putative DNA glycosylase are unknown. To test the potential UDG activity and evaluate phylogenetic classification, we isolated one SMUG1-like glycosylase representative from Listeria innocua (Lin). A biochemical screening of DNA glycosylase activity in vitro indicates that Lin SMUG1-like glycosylase is a single-strand selective uracil DNA glycosylase. The UDG activity on DNA bubble structures provides clue to its physiological significance in vivo. Mutagenesis and molecular modeling analyses reveal that Lin SMUG1-like glycosylase has similar functional motifs with SMUG1 enzymes; however, it contains a distinct catalytic doublet S67-S68 in motif 1 that is not found in any families in the UDG superfamily. Experimental investigation shows that the S67M-S68N double mutant is catalytically more active than either S67M or S68N single mutant. Coupled with mutual information analysis, the results indicate a high degree of correlation in the evolution of SMUG1-like enzymes. This study underscores the functional and catalytic diversity in the evolution of enzymes in UDG superfamily.     

Uracil-DNA glycosylase (UNG)-deficient mice reveal a primary role of the enzyme during DNA replication

Gene-targeted knockout mice have been generated lacking the major uracil-DNA glycosylase, UNG. In contrast to ung- mutants of bacteria and yeast, such mice do not exhibit a greatly increased spontaneous mutation frequency. However, there is only slow removal of uracil from misincorporated dUMP in isolated ung-/- nuclei and an elevated steady-state level of uracil in DNA in dividing ung-/- cells. A backup uracil-excising activity in tissue extracts from ung null mice, with properties indistinguishable from the mammalian SMUG1 DNA glycosylase, may account for the repair of premutagenic U:G mispairs resulting from cytosine deamination in vivo. The nuclear UNG protein has apparently evolved a specialized role in mammalian cells counteracting U:A base pairs formed by use of dUTP during DNA synthesis.     

Repair of Oxidative DNA Base Damage in the Host Genome Influences the HIV Integration Site Sequence Preference

Host base excision repair (BER) proteins that repair oxidative damage enhance HIV infection. These proteins include the oxidative DNA damage glycosylases 8-oxo-guanine DNA glycosylase (OGG1) and mutY homolog (MYH) as well as DNA polymerase beta (Polβ). While deletion of oxidative BER genes leads to decreased HIV infection and integration efficiency, the mechanism remains unknown. One hypothesis is that BER proteins repair the DNA gapped integration intermediate. An alternative hypothesis considers that the most common oxidative DNA base damages occur on guanines. The subtle consensus sequence preference at HIV integration sites includes multiple G:C base pairs surrounding the points of joining. These observations suggest a role for oxidative BER during integration targeting at the nucleotide level. We examined the hypothesis that BER repairs a gapped integration intermediate by measuring HIV infection efficiency in Polβ null cell lines complemented with active site point mutants of Polβ. A DNA synthesis defective mutant, but not a 5′dRP lyase mutant, rescued HIV infection efficiency to wild type levels; this suggeted Polβ DNA synthesis activity is not necessary while 5′dRP lyase activity is required for efficient HIV infection. An alternate hypothesis that BER events in the host genome influence HIV integration site selection was examined by sequencing integration sites in OGG1 and MYH null cells. In the absence of these 8-oxo-guanine specific glycosylases the chromatin elements of HIV integration site selection remain the same as in wild type cells. However, the HIV integration site sequence preference at G:C base pairs is altered at several positions in OGG1 and MYH null cells. Inefficient HIV infection in the absence of oxidative BER proteins does not appear related to repair of the gapped integration intermediate; instead oxidative damage repair may participate in HIV integration site preference at the sequence level.     

A novel uracil-DNA glycosylase family related to the helix-hairpin-helix DNA glycosylase superfamily

Cytosine bases can be deaminated spontaneously to uracil, causing DNA damage. Uracil-DNA glycosylase (UDG), a ubiquitous uracil-excising enzyme found in bacteria and eukaryotes, is one of the enzymes that repair this kind of DNA damage. To date, no UDG-coding gene has been identified in Methanococcus jannaschii, although its entire genome was deciphered. Here, we have identified and characterized a novel UDG from M.jannaschii designated as MjUDG. It efficiently removed uracil from both single- and double-stranded DNA. MjUDG also catalyzes the excision of 8-oxoguanine from DNA. MjUDG has a helix–hairpin–helix motif and a [4Fe–4S]-binding cluster that is considered to be important for the DNA binding and catalytic activity. Although MjUDG shares these features with other structural families such as endonuclease III and mismatch-specific DNA glycosylase (MIG), unique conserved amino acids and substrate specificity distinguish MjUDG from other families. Also, a homologous member of MjUDG was identified in Aquifex aeolicus. We report that MjUDG belongs to a novel UDG family that has not been described to date.     

DNA intermediates at the Escherichia coli replication fork. II. Studies using dut and ung mutants in vitro.

The incorporation of uracil into and excision from DNA were studied in vitro using lysates on cellophane discs made from Escherichia coli strains with defects in the enzymes dUTPase (dut) and uracil-DNA glycosylase (ung).Results with dut ung lysates indicate that dUTP is competitively incorporated with dTTP at the replication fork. Such incorporation is not due to DNA polymerase I. There is a mild discrimination (2.5-fold) against incorporation of dUTP versus dTTP. These data, together with in vivo uracil incorporation data (Tye et al., 1978) permit a rough estimate of the pool of dUTP in vivo (~0.5% of the dTTP pool).These in vitro data indicate that uracil-DNA glycosylase is the initial step in at least 90% of uracil excision events. However, in a strain defective in uracil-DNA glycosylase (ung-1), uracil-containing DNA is still more subject to single-strand scission than non-uracil-containing DNA, albeit at a rate at least tenfold less than in an ung⁺ strain.A number of qualitative statements may also be made about different steps in uracil incorporation and subsequent excision and repair events. When high levels of dUTP are added in vitro, a dut ung⁺ strain has a higher steady-state level of uracil in newly synthesized DNA than does an isogenic dut⁺ ung strain. Thus the dUTPase in these lysates has a higher capacity to be overloaded than does the excision system (i.e. uracil DNA glycosylase). However, the DNA sealing system (presumably DNA polymerase I and DNA ligase) apparently can handle all single-strand interruptions being introduced by uracil excision at the maximal rate, at least so that DNA synthesis can continue.