Mammalian 5-formyluracil-DNA glycosylase. 1. Identification and characterization of a novel activity that releases 5-formyluracil from DNA

5-Formyluracil (fU) is a major oxidative thymine lesion produced by reactive oxygen species and exhibits genotoxic and cytotoxic effects via several mechanisms. In the present study, we have searched for and characterized mammalian fU-DNA glycosylase (FDG) using two approaches. In the first approach, the FDG activity was examined using purified base excision repair enzymes. Human and mouse endonuclease III homologues (NTH1) showed a very weak FDG activity, but the parameter analysis and NaBH(4) trapping assays of the Schiff base intermediate revealed that NTH1 was kinetically incompetent for repair of fU. In the second approach, FDG was partially purified (160-fold) from rat liver. The enzyme was a monofunctional DNA glycosylase and recognized fU in single-stranded (ss) and double-stranded (ds) DNA. The most purified FDG fraction also exhibited monofunctional DNA glycosylase activities for uracil (U), 5-hydroxyuracil (hoU), and 5-hydroxymethyluracil (hmU) in ssDNA and dsDNA. The fU-excising activity of FDG was competitively inhibited by dsDNA containing U.G, hoU.G, and hmU.A but not by intact dsDNA containing T.A. Furthermore, the activities of FDG for fU, hmU, hoU, and U in ssDNA and dsDNA were neutralized by the antibody raised against SMUG1 uracil-DNA glycosylase, showing that FDG is a rat homologue of SMUG1.     

Substrate specificity of human endonuclease III (hNTH1). Effect of human APE1 on hNTH1 activity

Base excision repair of oxidized pyrimidines in human DNA is initiated by the DNA N-glycosylase/apurinic/apyrimidinic (AP) lyase, human NTH1 (hNTH1), the homolog of Escherichia coli endonuclease III (Nth). In contrast to Nth, the DNA N-glycosylase activity of hNTH1 is 7-fold greater than its AP lyase activity when the DNA substrate contains a thymine glycol (Tg) opposite adenine (Tg:A) (Marenstein, D. R., Ocampo, M. T. A., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2001) J. Biol. Chem. 276, 21242-21249). When Tg is opposite guanine (Tg:G), the two activities are of the same specific activity as the AP lyase activity of hNTH1 against Tg:A (Ocampo, M. T. A., Chaung, W., Marenstein, D. R., Chan, M. K., Altamirano, A., Basu, A. K., Boorstein, R. J., Cunningham, R. P., and Teebor, G. W. (2002) Mol. Cell. Biol. 22, 6111-6121). We demonstrate here that hNTH1 was inhibited by the product of its DNA N-glycosylase activity directed against Tg:G, the AP:G site. In contrast, hNTH1 was not as inhibited by the AP:A site arising from release of Tg from Tg:A. Addition of human APE1 (AP endonuclease-1) increased dissociation of hNTH1 from the DNA N-glycosylase-generated AP:A site, resulting in abrogation of AP lyase activity and an increase in turnover of the DNA N-glycosylase activity of hNTH1. Addition of APE1 did not abrogate hNTH1 AP lyase activity against Tg:G. The stimulatory protein YB-1 (Marenstein et al.), added to APE1, resulted in an additive increase in both activities of hNTH1 regardless of base pairing. Tg:A is formed by oxidative attack on thymine opposite adenine. Tg:G is formed by oxidative attack on 5-methylcytosine opposite guanine (Zuo, S., Boorstein, R. J., and Teebor, G. W. (1995) Nucleic Acids Res. 23, 3239-3243). It is possible that the in vitro substrate selectivity of mammalian NTH1 and the concomitant selective stimulation of activity by APE1 are indicative of selective repair of oxidative damage in different regions of the genome.     

5-Hydroxymethylcytosine DNA glycosylase activity in mammalian tissue

The enzymatic release of 5-hydroxymethylcytosine from T2 bacteriophage DNA was effected by an extract of calf thymus. Like the previously described 5-hydroxymethyluracil DNA glycosylase, 5-hydroxymethylcytosine DNA glycosylase was not detectable in bacterial extracts. The phylogenetic distribution of these activities indicates that their primary function is the maintenance of methylcytosine residues in differentiated tissue.     

Mammalian 5-formyluracil-DNA glycosylase. 2. Role of SMUG1 uracil-DNA glycosylase in repair of 5-formyluracil and other oxidized and deaminated base lesions

In the accompanying paper [Matsubara, M., et al. (2003) Biochemistry 42, 4993-5002], we have partially purified and characterized rat 5-formyluracil (fU)-DNA glycosylase (FDG). Several lines of evidence have indicated that FDG is a rat homologue of single-strand-selective monofunctional uracil-DNA glycosylase (SMUG1). We report here that rat and human SMUG1 (rSMUG1 and hSMUG1) expressed from the corresponding cDNAs indeed excise fU in single-stranded (ss) and double-stranded (ds) DNA. The enzymes also excised uracil (U) and uracil derivatives bearing an oxidized group at C5 [5-hydroxyuracil (hoU) and 5-hydroxymethyluracil (hmU)] in ssDNA and dsDNA but not analogous cytosine derivatives (5-hydroxycytosine and 5-formylcytosine) and other oxidized damage. The damage specificity and the salt concentration dependence of rSMUG1 (and hSMUG1) agreed well with those of FDG, confirming that FDG is rSMUG1. Consistent with the damage specificity above, hSMUG1 removed damaged bases from Fenton-oxidized calf thymus DNA, generating abasic sites. The amount of resulting abasic sites was about 10% of that generated by endonuclease III or 8-oxoguanine glycosylase in the same substrate. The HeLa cell extract and hSMUG1 exhibited a similar damage preference (hoU.G > hmU.A, fU.A), and the activities for fU, hmU, and hoU in the cell extract were effectively neutralized with hSMUG1 antibodies. These data indicate a dual role of hSMUG1 as a backup enzyme for UNG and a primary repair enzyme for a subset of oxidized pyrimidines such as fU, hmU, and hoU.     

Cellular effects of 5-formyluracil in DNA.

5-Formyluracil is a major oxidation product of thymine, formed in DNA in yields comparable to that of 8-oxo-7,8-dihydroguanine by exposure to gamma-irradiation. Whereas the repair pathways for removal and the biological effects of persisting 8-oxo-7,8-dihydroguanine are much elucidated, much less attention has been paid to the cellular implications of 5-formyluracil in DNA. Here we review the present state of knowledge in this important area within research on oxidative DNA damage.     

Oxanine DNA glycosylase activity from Mammalian alkyladenine glycosylase

Oxanine (Oxa) is a deaminated base lesion derived from guanine in which the N(1)-nitrogen is substituted by oxygen. This work reports the mutagenicity of oxanine as well as oxanine DNA glycosylase (ODG) activities in mammalian systems. Using human DNA polymerase beta, deoxyoxanosine triphosphate is only incorporated opposite cytosine (Cyt). When an oxanine base is in a DNA template, Cyt is efficiently incorporated opposite the template oxanine; however, adenine and thymine are also incorporated opposite Oxa with an efficiency approximately 80% of a Cyt/Oxa (C/O) base pair. Guanine is incorporated opposite Oxa with the least efficiency, 16% compared with cytosine. ODG activity was detected in several mammalian cell extracts. Among the known human DNA glycosylases tested, human alkyladenine glycosylase (AAG) shows ODG activity, whereas hOGG1, hNEIL1, or hNEIL2 did not. ODG activity was detected in spleen cell extracts of wild type age-matched mice, but little activity was observed in that of Aag knock-out mice, confirming that the ODG activity is intrinsic to AAG. Human AAG can excise Oxa from all four Oxa-containing double-stranded base pairs, Cyt/Oxa, Thy/Oxa, Ade/Oxa, and Gua/Oxa, with no preference to base pairing. Surprisingly, AAG can remove Oxa from single-stranded Oxa-containing DNA as well. Indeed, AAG can also remove 1,N(6)-ethenoadenine from single-stranded DNA. This study extends the deaminated base glycosylase activities of AAG to oxanine; thus, AAG is a mammalian enzyme that can act on all three purine deamination bases, hypoxanthine, xanthine, and oxanine.     

Stimulation of DNA glycosylase activity of OGG1 by NEIL1: functional collaboration between two human DNA glycosylases

The eukaryotic 8-oxoguanine-DNA glycosylase 1 (OGG1) provides the major activity for repairing mutagenic 7,8-dihydro-8-oxoguanine (8-oxoG) induced in the genome due to oxidative stress. Earlier in vitro studies showed that, after excising the base lesion, the human OGG1 remains bound to the resulting abasic (AP) site in DNA and does not turn over efficiently. The human AP-endonuclease (APE1), which cleaves the phosphodiester bond 5' to the AP site, in the next step of repair, displaces the bound OGG1 and thus increases its turnover. Here we show that NEIL1, a DNA glycosylase/AP lyase specific for many oxidized bases but with weak 8-oxoG excision activity, stimulates turnover of OGG1 in a fashion similar to that of APE1 and carries out betadelta-elimination at the AP site. This novel collaboration of two DNA glycosylases, which do not stably interact with each other, in stimulating 8-oxoguanine repair is possible because of higher AP site affinity and stronger AP lyase activity of NEIL1 relative to OGG1. Comparable levels of NEIL1 and OGG1 in some human cells raise the possibility that NEIL1 serves as a backup enzyme to APE1 in stimulating 8-oxoG repair in vivo.     

Excision of 5-halogenated uracils by human thymine DNA glycosylase. Robust activity for DNA contexts other than CpG

Thymine DNA glycosylase (TDG) excises thymine from G.T mispairs and removes a variety of damaged bases (X) with a preference for lesions in a CpG.X context. We recently reported that human TDG rapidly excises 5-halogenated uracils, exhibiting much greater activity for CpG.FU, CpG.ClU, and CpG.BrU than for CpG.T. Here we examine the effects of altering the CpG context on the excision activity for U, T, FU, ClU, and BrU. We show that the maximal activity (k(max)) for G.X substrates depends significantly on the 5' base pair. For example, k(max) decreases by 6-, 11-, and 82-fold for TpG.ClU, GpG.ClU, and ApG.ClU, respectively, as compared with CpG.ClU. For the other G.X substrates, the 5'-neighbor effects have a similar trend but vary in magnitude. The activity for G.FU, G.ClU, and G.BrU, with any 5'-flanking pair, meets and in most cases significantly exceeds the CpG.T activity. Strikingly, human TDG activity is reduced 10(2.3)-10(4.3)-fold for A.X relative to G.X pairs and reduced further for A.X pairs with a 5' pair other than C.G. The effect of altering the 5' pair and/or the opposing base (G.X versus A.X) is greater for substrates that are larger (bromodeoxyuridine, dT) or have a more stable N-glycosidic bond (such as dT). The largest CpG context effects are observed for the excision of thymine. The potential role played by human TDG in the cytotoxic effects of ClU and BrU incorporation into DNA, which can occur under inflammatory conditions and in the cytotoxicity of FU, a widely used anticancer agent, are discussed.     

Truncation of amino-terminal tail stimulates activity of human endonuclease III (hNTH1)

Human MRP14 (hMRP14) is a Ca(2+)-binding protein from the S100 family of proteins. This protein is co-expressed with human MRP8 (hMRP8), a homologue protein in myeloid cells, and plays an indispensable role in Ca(2+)-dependent functions during inflammation. This role includes the activation of Mac-1, the beta(2) integrin which is involved in neutrophil adhesion to endothelial cells. The crystal structure of the holo form of hMRP14 was analyzed at 2.1 A resolution. hMRP14 is distinguished from other S100 member proteins by its long C-terminal region, and its structure shows that the region is extensively flexible. In this crystal structure of hMRP14, Chaps molecules bind to the hinge region that connects two EF-hand motifs, which suggests that this region is a target-binding site of this protein. Based on a structural comparison of hMRP14 with hMRP8 and human S100A12 (hS100A12) that is another homologue protein, the character of MRP8/14 hetero-complex and the functional significance of the flexibility of the C-terminal region of hMRP14 are discussed.     

The human Werner syndrome protein stimulates repair of oxidative DNA base damage by the DNA glycosylase NEIL1

The mammalian DNA glycosylase, NEIL1, specific for repair of oxidatively damaged bases in the genome via the base excision repair pathway, is activated by reactive oxygen species and prevents toxicity due to radiation. We show here that the Werner syndrome protein (WRN), a member of the RecQ family of DNA helicases, associates with NEIL1 in the early damage-sensing step of base excision repair. WRN stimulates NEIL1 in excision of oxidative lesions from bubble DNA substrates. The binary interaction between NEIL1 and WRN (K(D) = 60 nM) involves C-terminal residues 288-349 of NEIL1 and the RecQ C-terminal (RQC) region of WRN, and is independent of the helicase activity WRN. Exposure to oxidative stress enhances the NEIL-WRN association concomitant with their strong nuclear co-localization. WRN-depleted cells accumulate some prototypical oxidized bases (e.g. 8-oxoguanine, FapyG, and FapyA) indicating a physiological function of WRN in oxidative damage repair in mammalian genomes. Interestingly, WRN deficiency does not have an additive effect on in vivo damage accumulation in NEIL1 knockdown cells suggesting that WRN participates in the same repair pathway as NEIL1.     

Thymine DNA glycosylase specifically recognizes 5-carboxylcytosine-modified DNA

Human thymine DNA glycosylase (hTDG) efficiently excises 5-carboxylcytosine (5caC), a key oxidation product of 5-methylcytosine in genomic DNA, in a recently discovered cytosine demethylation pathway. We present here the crystal structures of the hTDG catalytic domain in complex with duplex DNA containing either 5caC or a fluorinated analog. These structures, together with biochemical and computational analyses, reveal that 5caC is specifically recognized in the active site of hTDG, supporting the role of TDG in mammalian 5-methylcytosine demethylation.     

Structural characterization of viral ortholog of human DNA glycosylase NEIL1 bound to thymine glycol or 5-hydroxyuracil-containing DNA

Thymine glycol (Tg) and 5-hydroxyuracil (5-OHU) are common oxidized products of pyrimidines, which are recognized and cleaved by two DNA glycosylases of the base excision repair pathway, endonuclease III (Nth) and endonuclease VIII (Nei). Although there are several structures of Nei enzymes unliganded or bound to an abasic (apurinic or apyrimidinic) site, until now there was no structure of an Nei bound to a DNA lesion. Mimivirus Nei1 (MvNei1) is an ortholog of human NEIL1, which was previously crystallized bound to DNA containing an apurinic site (Imamura, K., Wallace, S. S., and Doublié, S. (2009) J. Biol. Chem. 284, 26174-26183). Here, we present two crystal structures of MvNei1 bound to two oxidized pyrimidines, Tg and 5-OHU. Both lesions are flipped out from the DNA helix. Tg is in the anti conformation, whereas 5-OHU adopts both anti and syn conformations in the glycosylase active site. Only two protein side chains (Glu-6 and Tyr-253) are within hydrogen-bonding contact with either damaged base, and mutating these residues did not markedly affect the glycosylase activity. This finding suggests that lesion recognition by Nei occurs before the damaged base flips into the glycosylase active site.     

Functionality of human thymine DNA glycosylase requires SUMO-regulated changes in protein conformation

BACKGROUND: Base excision repair initiated by human thymine-DNA glycosylase (TDG) results in the generation of abasic sites (AP sites) in DNA. TDG remains bound to this unstable repair intermediate, indicating that its transmission to the downstream-acting AP endonuclease is a coordinated process. Previously, we established that posttranslational modification of TDG with Small Ubiquitin-like MOdifiers (SUMOs) facilitates the dissociation of the DNA glycosylase from the product AP site, but the underlying molecular mechanism remained unclear. RESULTS: We now show that upon DNA interaction, TDG undergoes a dramatic conformational change, which involves its flexible N-terminal domain and accounts for the nonspecific DNA binding ability of the enzyme. This function is required for efficient processing of the G.T mismatch but then cooperates with the specific DNA contacts established in the active site pocket of TDG to prevent its dissociation from the product AP site after base release. SUMO1 conjugation to the C-terminal K330 of TDG modulates the DNA binding function of the N terminus to induce dissociation of the glycosylase from the AP site while it leaves the catalytic properties of base release in the active site pocket of the enzyme unaffected. CONCLUSION: Our data provide insight into the molecular mechanism of SUMO modification mediated modulation of enzymatic properties of TDG. A conformational change, involving the N-terminal domain of TDG, provides unspecific DNA interactions that facilitate processing of a wider spectrum of substrates at the expense of enzymatic turnover. SUMOylation then reverses this structural change in the product bound TDG.     

The C-terminal alphaO helix of human Ogg1 is essential for 8-oxoguanine DNA glycosylase activity: the mitochondrial beta-Ogg1 lacks this domain and does not have glycosylase activity

The human Ogg1 glycosylase is responsible for repairing 8-oxo-7,8-dihydroguanine (8-oxoG) in both nuclear and mitochondrial DNA. Two distinct Ogg1 isoforms are present; α-Ogg1, which mainly localizes to the nucleus and β-Ogg1, which localizes only to mitochondria. We recently showed that mitochondria from ρ⁰ cells, which lack mitochondrial DNA, have similar 8-oxoG DNA glycosylase activity to that of wild-type cells. Here, we show that β-Ogg1 protein levels are ~80% reduced in ρ⁰ cells, suggesting β-Ogg1 is not responsible for 8-oxoG incision in mitochondria. Thus, we characterized the biochemical properties of recombinant β-Ogg1. Surprisingly, recombinant β-Ogg1 did not show any significant 8-oxoG DNA glycosylase activity in vitro. Since β-Ogg1 lacks the C-terminal αO helix present in α-Ogg1, we generated mutant proteins with various amino acid substitutions in this domain. Of the seven amino acid positions substituted (317–323), we identified Val-317 as a novel critical residue for 8-oxoG binding and incision. Our results suggest that the αO helix is absolutely necessary for 8-oxoG DNA glycosylase activity, and thus its absence may explain why β-Ogg1 does not catalyze 8-oxoG incision in vitro. Western blot analysis revealed the presence of significant amounts of α-Ogg1 in human mitochondria. Together with previous localization studies in vivo, this suggests that α-Ogg1 protein may provide the 8-oxoG DNA glycosylase activity for the repair of these lesions in human mitochondrial DNA. β-Ogg1 may play a novel role in human mitochondria.     

Identification and characterization of a novel human DNA glycosylase for repair of cytosine-derived lesions

Two candidate human orthologs of Escherichia coli MutM/Nei were recently identified in the human genome database, and one of these, NEH1, was characterized earlier (Hazra, T. K., Izumi, T., Boldogh, I., Imhoff, B., Kow, Y. W., Jaruga, P., and Dizdaroglu, M. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3523-3528). Here we report characterization of the second protein, originally named NEH2 and now renamed NEIL2 (Nei-like). The 37-kDa wild-type NEIL2 expressed in and purified from E. coli has DNA glycosylase/AP lyase activity, primarily for excising oxidative products of cytosine, with highest activity for 5-hydroxyuracil, one of the most abundant and mutagenic lesions induced by reactive oxygen species, and with lower activity for 5,6-dihydrouracil and 5-hydroxycytosine. It has negligible or undetectable activity with 8-oxoguanine, thymine glycol, 2-hydroxyadenine, hypoxanthine, and xanthine. NEIL2 is similar to NEIL1 in having N-terminal Pro as the active site. However, unlike NEIL1, its expression was independent of the cell cycle stage in fibroblasts, and its highest expression was observed in the testes and skeletal muscle. Despite the absence of a putative nuclear localization signal, NEIL2 was predominantly localized in the nucleus. These results suggest that NEIL2 is involved in global genome repair mainly for removing oxidative products of cytosine.     

Estradiol receptor potentiates,in vitro,the activity of 5-methylcytosine DNA glycosylase

Heme oxygenase-1 (HO-1) is induced under various oxidative stress conditions, such as lipopolysaccharide (LPS) insult. Induction of HO-1 by LPS is reported to be mediated through interleukin-1beta (IL-1beta), rather than other inflammatory cytokines in the mouse liver. However, we found that IL-1alpha/beta knockout (KO) mice responded well to LPS insult, as did wild-type mice with respect to HO-1 mRNA induction (about 30-fold increase). In contrast, tumor necrosis factor alpha KO (TNFalphaKO) mice responded very weakly to LPS in the HO-1 mRNA expression, but not metallothionein mRNA. Recent studies reveal that nitric oxide from Kupffer cells is involved in HO-1 induction in the liver produced by LPS. Therefore, nitrite and nitrate concentrations in the liver were also measured and these parameters did not increase in either IL-1KO or TNFalphaKO. In addition, the phosphorylation of c-JUN N-terminal kinase (JNK) and p38, but not extracellular signal-regulated kinase, was very low in TNFalphaKO mice due to LPS administration. All of these findings indicate that TNFalpha is a major candidate to trigger HO-1 induction in response to LPS stimulation, and that its message is likely transduced through JNK and p38 pathways.     

Biochemical mapping of human NEIL1 DNA glycosylase and AP lyase activities

Base excision repair of oxidized DNA in human cells is initiated by several DNA glycosylases with overlapping substrate specificity. The human endonuclease VIII homologue NEIL1 removes a broad spectrum of oxidized pyrimidine and purine lesions. In this study of NEIL1 we have identified several key residues, located in three loops lining the DNA binding cavity, important for lesion recognition and DNA glycosylase/AP lyase activity for oxidized bases in double-stranded and single-stranded DNA. Single-turnover kinetics of NEIL1 revealed that removal of 5-hydroxycytosine (5-OHC) and 5-hydroxyuracil (5-OHU) is ～25 and ～10-fold faster in duplex DNA compared to single-stranded DNA, respectively, and also faster than removal of dihydrothymine (DHT) and dihydrouracil (DHU), both in double-stranded and single-stranded DNA. NEIL1 excised 8-oxoguanine (8-oxoG) only from double-stranded DNA and analysis of site-specific mutants revealed that Met81, Arg119 and Phe120 are essential for removal of 8-oxoG. Further, several arginine and histidine residues located in the loop connecting the two β-strands forming the zincless finger motif and projecting into the DNA major groove, were shown to be imperative for lesion processing for both single- and double-stranded substrates. Trapping experiments of active site mutants revealed that the N-terminal Pro2 and Lys54 can alternate to form a Schiff-base complex between the protein and DNA. Hence, both Pro2 and Lys54 are involved in the AP lyase activity. While wildtype NEIL1 activity almost exclusively generated a δ-elimination product when processing single-stranded substrates, substitution of Lys54 changed this in favor of a β-elimination product. These results suggest that Pro2 and Lys54 are both essential for the concerted action of the β,δ-elimination in NEIL1.     

5-Methylcytosine DNA glycosylase activity is also present in the human MBD4 (G/T mismatch glycosylase) and in a related avian sequence

A 1468 bp cDNA coding for the chicken homolog of the human MBD4 G/T mismatch DNA glycosylase was isolated and sequenced. The derived amino acid sequence (416 amino acids) shows 46% identity with the human MBD4 and the conserved catalytic region at the C-terminal end (170 amino acids) has 90% identity. The non-conserved region of the avian protein has no consensus sequence for the methylated DNA binding domain. The recombinant proteins from human and chicken have G/T mismatch as well as 5-methylcytosine (5-MeC) DNA glycosylase activities. When tested by gel shift assays, human recombinant protein with or without the methylated DNA binding domain binds equally well to symmetrically, hemimethylated DNA and non-methylated DNA. However, the enzyme has only 5-MeC DNA glycosylase activity with the hemimethylated DNA. Footprinting of human MBD4 and of an N-terminal deletion mutant with partially depurinated and depyrimidinated substrate reveal a selective binding of the proteins to the modified substrate around the CpG. As for 5-MeC DNA glycosylase purified from chicken embryos, MBD4 does not use oligonucleotides containing mCpA, mCpT or mCpC as substrates. An mCpG within an A+T-rich oligonucleotide is a much better substrate than an A+T-poor sequence. The K_m of human MBD4 for hemimethylated DNA is ~10^–7 M with a V_max of ~10^–11 mol/h/µg protein. Deletion mutations show that G/T mismatch and 5-MeC DNA glycosylase are located in the C-terminal conserved region. In sharp contrast to the 5-MeC DNA glycosylase isolated from the chicken embryo DNA demethylation complex, the two enzymatic activities of MBD4 are strongly inhibited by RNA. In situ hybridization with antisense RNA indicate that MBD4 is only located in dividing cells of differentiating embryonic tissues.