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• W2068407072 abstract "Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (β-δ) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression. Thymine glycol, a potentially lethal DNA lesion produced by reactive oxygen species, can be removed by DNA glycosylase, Escherichia coli Nth (endonuclease III), or its mammalian homologue NTH1. We have found previously that mice deleted in the Nth homologue still retain at least two residual glycosylase activities for thymine glycol. We report herein that in cell extracts from the mNth1 knock-out mouse there is a third thymine glycol glycosylase activity that is encoded by one of three mammalian proteins with sequence similarity to E. coli Fpg (MutM) and Nei (endonuclease VIII). Tissue expression of this mouse Nei-like (designated as Neil1) gene is ubiquitous but much lower than that of mNth1 except in heart, spleen, and skeletal muscle. Recombinant NEIL1 can remove thymine glycol and 5-hydroxyuracil in double- and single-stranded DNA much more efficiently than 8-oxoguanine and can nick the strand by an associated (β-δ) apurinic/apyrimidinic lyase activity. In addition, the mouse NEIL1 has a unique DNA glycosylase/lyase activity toward mismatched uracil and thymine, especially in U:C and T:C mismatches. These results suggest that NEIL1 is a back-up glycosylase for NTH1 with unique substrate specificity and tissue-specific expression. 8-oxoguanine apurinic/apyrimidinic thymine glycol base excision repair 5-hydroxyuracil nickel-nitrilotriacetic acid high pressure liquid chromatography helix-two-turn-helix transcription-coupled repair Oxidative damage in DNA is widely acknowledged to be a causative factor in cancer and aging (1Ames B.N. Shigenaga M.K. Ann. N. Y. Acad. Sci. 1992; 663: 85-96Crossref PubMed Scopus (265) Google Scholar, 2Beckman K.B. Ames B.N. J. Biol. Chem. 1997; 272: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (848) Google Scholar). Major oxidative damage includes premutagenic lesions and lesions that block replicative DNA polymerases. A typical example of a premutagenic lesion is 8-oxoguanine (8-oxoG),1 which can form a base pair with adenine, as well as with cytosine, resulting in G:C to T:A transversion. In contrast to 8-oxoG, a major replication-blocking lesion generated by reactive oxygen species in vivo is thymine glycol (Tg). Therefore, both 8-oxoG and Tg are often used as biomarkers for oxidative stress and aging (3Ames B.N. Mutat. Res. 1989; 214: 41-46Crossref PubMed Scopus (291) Google Scholar, 4Le X.C. Xing J.Z. Lee J. Leadon S.A. Weinfeld M. Science. 1998; 280: 1066-1069Crossref PubMed Scopus (187) Google Scholar). Despite the importance of Tg, repair pathway of Tg in mammalian cells is not well understood. It has been well established that the most important repair pathway for the removal of mutagenic or toxic oxidative base damage is base excision repair (BER). The marked evolutionary conservation of BER enzymes from bacteria to mammals (5Memisoglu A. Samson L. Mutat. Res. 2000; 451: 39-51Crossref PubMed Scopus (239) Google Scholar, 6Nilsen H. Krokan H.E. Carcinogenesis. 2001; 22: 987-998Crossref PubMed Scopus (178) Google Scholar) suggests the importance of BER for life and has enabled the identification of various mammalian homologues through database searches using the nucleotide sequences encoding well characterized Escherichia coli and yeast enzymes. Three mammalian genes for oxidative DNA repair glycosylases, OGG1, MYH, and NTH1, have been identified in this way. In addition, the growing amount of information provided by genome and cDNA sequencing projects has elucidated species-specific differences between enzymes involved in the conserved BER reaction. Prokaryotes employ Fpg glycosylase for repair of oxidized purines, whereas eukaryotes use OGG1, which has almost no amino acid sequence similarity with the bacterial Fpg protein. A homologue of OGG1 is also found in archaea (7Gogos A. Clarke N.D. J. Biol. Chem. 1999; 274: 30447-30450Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar), whereas Arabidopsis thaliana has both Fpg and OGG1 (8Garcia-Ortiz M.V. Ariza R.R. Roldan-Arjona T. Plant Mol. Biol. 2001; 47: 795-804Crossref PubMed Scopus (58) Google Scholar). E. coli possesses two DNA glycosylases for repair of oxidized pyrimidine, Nth and Nei, which are structurally unrelated but functional homologues. nth/nei double-deficient bacteria, but not the single mutant, show sensitivity to hydrogen peroxide and ionizing radiation (9Jiang D. Hatahet Z. Blaisdell J.O. Melamede R.J. Wallace S.S. J. Bacteriol. 1997; 179: 3773-3782Crossref PubMed Google Scholar, 10Saito Y. Uraki F. Nakajima S. Asaeda A. Ono K. Kubo K. Yamamoto K. J. Bacteriol. 1997; 179: 3783-3785Crossref PubMed Scopus (91) Google Scholar). Nth is one of the most widespread DNA glycosylases in eukaryotes, as well as prokaryotes, whereas the Nei orthologs are rarely found in bacteria and are even absent in theSaccharomyces cerevisiae, Schizosaccharomyces pombe, and Drosophila melanogaster genomes. Although this distribution suggests that mammalian species may also lack Nei homologues, three Fpg/Nei DNA glycosylase-like sequences have recently been registered in a full-length cDNA database (NEDO human cDNA sequencing project; www.nedo.go.jp/bio/). We have characterized these Fpg Nei-Like genes and their repair activities in recombinant proteins. During preparation and submission of this manuscript, these clones have been reported (11Hazra T.K. Izumi T. Boldogh I. Imhoff B. Kow Y.W. Jaruga P. Dizdaroglu M. Mitra S. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3523-3528Crossref PubMed Scopus (440) Google Scholar, 12Bandaru V. Sunkara S. Wallace S.S. Bond J.P. DNA Repair. 2002; 1: 517-529Crossref PubMed Scopus (286) Google Scholar, 13Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar) and, accordingly, we use the name NEIL (Nei-Like) for the genes in this paper. We have recently established Nth1 knock-out mice to investigate the biological consequences of oxidative pyrimidine repair deficiency (14Takao M. Kanno S. Shiromoto T. Hasegawa R. Ide H. Ikeda S. Sarker A.H. Seki S. Xing J.X., Le, C. Weinfeld M. Kobaashi K. Miyazaki J. Muijtjens M. Hoeijmakers J.H.J. van der Horst G. Yasui A. EMBO J. 2002; 21: 3486-3493Crossref PubMed Scopus (139) Google Scholar). Surprisingly, the NTH1-deficient mice lack overt phenotypic abnormalities. Moreover, Tg produced in the mutant mouse liver DNA by X-irradiation disappeared with time, though more slowly than in the wild-type mouse. Biochemical analysis of NTH1-deficient mouse liver extracts showed that there are at least two Tg-DNA glycosylase back-up activities. One activity designated as TGG1 (thymine glycol glycosylase 1) is a monofunctional DNA glycosylase found mainly in the mitochondria, and the other TGG2 forms a Schiff base reaction intermediate, which is indicative of a nuclear AP lyase-associating enzyme. Because we found three human NEIL genes in the database, we analyzed the enzymatic activity of the proteins in vitro and in vivo and their relationships to residual glycosylase activities inmNth1 knock-out mice. Primers for the PCR were designed based on the cDNA sequences in GenBankTM(AK026055 for FLJ22402/hNEIL1, AK056206 for FLJ31644, AK001720 for FLJ10858, and AK013322 for mouse ortholog of FLJ22402/mNEIL1). The cDNA for human FLJ31644, FLJ10858, or mNEIL1 was amplified from a cDNA pool reverse-transcribed from mRNA from HeLa cells, keratinocytes, or NTH1-deficient mouse liver, respectively. The cDNA for hNEIL1 was amplified from a commercial human testis cDNA library (Invitrogen). The cDNA was synthesized with a kit using oligo(dT) primers (Roche Molecular Biochemicals). The amplified fragment was subcloned in pSTBlue-1 (Novagen). Multiple clones obtained were sequenced extensively to exclude mutants generated by PCR. The sequence of any reamplified fragment was also verified. These sequence data have been submitted to the DDBJ/GenBankTM/EBI database under accession number AB079068 (human NEIL1), AB079069 (mouse NEIL1/FLJ22402), AB079070 (human NEIL2/FLJ31644), and AB079071 (human NEIL3/FLJ10858). The initial PCR mentioned above was made with a 5′-primer containing Kozak's sequence (ACC) in front of the initiation codon. The cDNA fragment was subcloned into a pSPUTK vector (Stratagene). In vitroexpression was performed with a Quick TNT system (Promega) in the presence of [35S]methionine for the detection of protein production or cold methionine for activity screening. The translated products were analyzed on SDS-PAGE with a 10–20% gradient gel (Daiichi Pure Chemicals). Quantitative PCR was performed using a LightCycler (Roche Molecular Biochemicals) with a DNA Master SYBR Green I kit (Roche Molecular Biochemicals) according to the system and kit instructions. A mouse multiple tissue cDNA panel was purchased from Clontech. Amplimers used were as follows: mouse glyceraldehyde-3-phosphate dehydrogenase gene (Gapdh) primers, 5′-CCTTGGCTCCTGCATGTGCCCAAT and 5′-CATGTAGGCCATGAGGTCCACCAC; Nth1 primers, 5′-GCCACAGGCCCGTGAGACATCCACGGAGAA and 5′-GCACTTGCATCATAGCAGTGCTCG; Neil1 primers, 5′-CTGACCCTGAGCCAGAAGATCAA and 5′-CCCAGCTGTGTCTCCTGTGACTT. Standard curves were obtained by quantitative PCR with a known amount of linearized plasmid DNA containing the respective cDNA. Prior to quantification, the cDNA panel was re-equalized by PCR for Gapdh. Data were expressed relative to the expression of Gapdh. An epitope tag (FLAG sequence) was added to mNeil1 cDNA by PCR. The tagged cDNA was subcloned in a mammalian expression vector pTargeT (Promega). COS-7 cells were transfected with the construct using FuGENE6 (Roche Molecular Biochemicals). The transiently expressed protein was detected by an anti-FLAG antibody (Sigma-Aldrich) and an Alexa Fluor 488-conjugated second antibody (Molecular Probes). The incision assay was made with a 30-mer oligonucleotide containing a single modified base indicated byX, 5′-CTCGTCAGCATCTXCATCATACAGTCAGTG-3′. Oligos containing Tg, 8-oxoG, and the tetrahydrofuran AP site (fAP) were generously provided by Dr. S. Iwai (Tokyo University). Oligos containing 5-hydroxyuracil (5-OHU) and uracil were synthesized and purified by Japan Bio-Service. The oligo was labeled using either T4-polynucleotide kinase (TaKaRa) with [γ-32P]ATP (5000 Ci/mmol; Amersham Biosciences) or terminal deoxynucleotidyl transferase (Invitrogen) with [α-32P]ddATP (5000 Ci/mmol; Amersham Biosciences). The incision reaction was performed in reaction buffer (20 mm sodium phosphate (pH 7.5), 50 mm NaCl, 5 mm EDTA, and 100 μg/ml bovine serum albumin) containing 1 fmol/μl of either double-stranded DNA or single-stranded DNA at 30 °C for 30 min. The reaction was stopped by addition of an equal volume of 90% formamide loading dye solution. The samples were heated at 75 °C for 2 min and run on a 20% polyacrylamide gel containing 7m urea. The gel was exposed to an imaging plate and analyzed with a BAS2000 image analyzer (Fuji photo film). The densitometric analysis of the gel image was performed with NIH Image software. For the reaction with in vitro translated gene products, the reacted DNA was hydrolyzed with alkaline as described previously (15Takao M. Zhang Q.-M. Yonei S. Yasui A. Nucleic Acids Res. 1999; 27: 3638-3644Crossref PubMed Scopus (151) Google Scholar). Briefly, the DNA was phenol/chloroform-extracted and ethanol-precipitated. The DNA was dissolved in 0.1 m NaOH and incubated at 95 °C for 5 min to convert the abasic DNA into the nicked form. In some experiments, we used T4 endonuclease V as “AP lyase” enzyme for detecting monofunctional DNA glycosylase, as well as for assays with crude extract (14Takao M. Kanno S. Shiromoto T. Hasegawa R. Ide H. Ikeda S. Sarker A.H. Seki S. Xing J.X., Le, C. Weinfeld M. Kobaashi K. Miyazaki J. Muijtjens M. Hoeijmakers J.H.J. van der Horst G. Yasui A. EMBO J. 2002; 21: 3486-3493Crossref PubMed Scopus (139) Google Scholar). To characterize the terminal structure of the incision products, 3′-labeled or 5′-labeled DNA digests were treated with bacterial alkaline phosphatase (TaKaRa) or with T4 polynucleotide kinase in a buffer containing 20 mm sodium phosphate (pH 6.0), 10 mmMgCl2, 5 mm 2-mercaptoethanol, respectively (16Cameron V. Uhlenbeck O.C. Biochemistry. 1977; 16: 5120-5126Crossref PubMed Scopus (227) Google Scholar). A His6-tagged expression construct was made by subcloningmNeil1 into pET21 vector (Novagen). The E. coliBL21 codon plus (Stratagene) was transformed and cultured at 37 °C until A 600 reached 0.6. After induction with 0.5 mm isopropyl-1-thio-β-d-galactopyranoside at 25 °C for 6 h, Ni-NTA (Qiagen) column chromatography was performed under native conditions according to the manufacturer's instructions. The recombinant NEIL1 protein was eluted in a 20 mm sodium phosphate buffer (pH 8.0) containing 100 mm NaCl and 250 mm imidazole. The eluate was directly loaded onto HiTrap-SP (Amersham Biosciences), and the active fractions eluted in phosphate buffer containing 0.5–0.7 M NaCl were pooled. The purified NEIL1 protein was concentrated to 1 mg/ml and stored in 25 mm sodium phosphate buffer (pH 8.0), 50 mm NaCl, 1 mm dithiothreitol, 1 mmEDTA, and 50% glycerol at −20 °C. Because the activity gradually decreased, the quantitative and comparative experiments were made with the same purification lot within 2 days. For DNA trapping, the recombinant NEIL1 protein or column fraction of mouse cell extracts was mixed with 100 mm NaCNBH3 in 50 mm sodium phosphate buffer (pH 7.5) containing 10 mm EDTA on ice. Labeled Tg-containing oligo (final concentration, 5 fmol/μl) was added and incubated at 30 °C for 60 min. The 50-μl reaction mix was applied on a Sephadex G-50 microcolumn (Amersham Biosciences) to remove the reductant. The enzyme-DNA complex was separated on a 10–20% SDS-polyacrylamide gel and autoradiographed. For limited proteolysis experiments, a portion of the G-50 gel-filtrated sample was digested further either by trypsin or chymotrypsin. Partial purification of Tg-DNA glycosylase activities from NTH1-deficient mouse liver nuclear extracts has been described in a previous paper (14Takao M. Kanno S. Shiromoto T. Hasegawa R. Ide H. Ikeda S. Sarker A.H. Seki S. Xing J.X., Le, C. Weinfeld M. Kobaashi K. Miyazaki J. Muijtjens M. Hoeijmakers J.H.J. van der Horst G. Yasui A. EMBO J. 2002; 21: 3486-3493Crossref PubMed Scopus (139) Google Scholar). In brief, nuclear proteins were extracted in Buffer A (50 mm Tris-HCl (pH 7.5), 0.1% Nonidet P-40, 2 mm dithiothreitol) with 0.3 mNaCl. The supernatant was dialyzed in Buffer B (50 mmTris-HCl (pH 7.5), 2 mm dithiothreitol. 1 mmEDTA, 10% glycerol) with 0.1 m NaCl and applied to heparin-agarose and eluted with Buffer B containing 0.8 mNaCl. The fraction was dialyzed and applied to an UNO S-1 column in an HPLC system (Bio-Rad). Incision assays and trapping assays were conducted on the flow-through fraction, the fractions separated by a linear gradient of 0.1–0.6 m NaCl, and the fraction eluted by stepwise increase of NaCl from 0.6 to 1.0 m. A BLAST homology search of GenBankTM using theE. coli Nei sequence as query yielded three human sequences for hypothetical proteins (FLJ22402, FLJ31644, and FLJ10858) that exhibit significant similarity to E. coli Fpg and Nei (Fig.1). From this similarity we designate these Fpg-Nei-like proteins as follows (13Hazra T.K. Kow Y.W. Hatahet Z. Imhoff B. Boldogh I. Mokkapati S.K. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: 30417-30420Abstract Full Text Full Text PDF PubMed Scopus (286) Google Scholar): NEIL1 for FLJ22402, NEIL2 for FLJ31644, and NEIL3 for FLJ10858. The human NEIL1, NEIL2, and NEIL3 genes map on chromosomes 15, 8, and 4, respectively. The respective cDNA structure and approximate positions of introns inferred from its genomic sequence are shown in Fig. 1 B. The crystal structures of Thermus thermophilus Fpg and Nei show a significant homology (17Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar, 18Zharkov D.O. Rieger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-5341Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar), delineating a DNA binding cleft composed of common structural elements and conserved amino acid residues. The structural analysis suggests further that a protein potentially belonging to the Fpg-Nei protein family has six conserved DNA-contacting elements in its sequence: the N-terminal active site, turn β3-β4, turn β5-β6, turn β8-β9, helix-two-turn-helix (H2TH) motif, and zinc-finger motif (numbering of the β-sheets refers to Tth-Fpg structure; see Ref. 17Sugahara M. Mikawa T. Kumasaka T. Yamamoto M. Kato R. Fukuyama K. Inoue Y. Kuramitsu S. EMBO J. 2000; 19: 3857-3869Crossref PubMed Scopus (138) Google Scholar). Indeed, these domains are highly conserved among the known and putative prokaryotic Fpg and Nei sequences in the database. To assess a possible DNA binding capacity of the human proteins, we searched for the DNA-contacting elements and re-aligned the NEIL sequences with Fpg or Nei (Fig. 1 A). All three proteins contain a H2TH motif, representing a helix-hairpin-helix motif widespread in DNA-binding proteins including many DNA glycosylases (19Shao X. Grishin N.V. Nucleic Acids Res. 2000; 28: 2643-2650Crossref PubMed Scopus (113) Google Scholar). The prototype helix-hairpin-helix motif has a consensus GhG (h is a hydrophobic residue) sequence between the two helices that is found in all three proteins. Moreover, the three NEIL proteins have absolutely conserved residues (Asp, Asn, and Glu) specific for the H2TH motif of the Fpg-Nei family. The NEIL1 and NEIL2 proteins contain the conserved N-terminal active site residues, Pro-2 and Glu-3, whereas in the NEIL3 protein the Schiff base-forming Pro-2 (18Zharkov D.O. Rieger R.A. Iden C.R. Grollman A.P. J. Biol. Chem. 1997; 272: 5335-5341Abstract Full Text Full Text PDF PubMed Scopus (176) Google Scholar, 20Rieger R.A. McTigue M.M. Kycia J.H. Gerchman S.E. Grollman A.P. Iden C.R. J. Am. Soc. Mass Spectrom. 2000; 11: 505-515Crossref PubMed Scopus (48) Google Scholar, 21Sidorkina O.M. Laval J. J. Biol. Chem. 2000; 275: 9924-9929Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar) is replaced by valine. The zinc-finger motif is conserved in NEIL3 and to a lesser extent in NEIL2, whereas both retain the invariable Arg and Gln at the appropriate positions. Interestingly, the NEIL1 protein does not show any homology to Fpg or Nei in the C-terminal region of the H2TH motif. Nevertheless, the NEIL1 sequence alone exhibits all the three DNA-contacting turns (β3-β4, β5-β6, and β8-β9) with great resemblance to Fpg or Nei. The relative position of these potential DNA-contacting elements is depicted schematically in the respective cDNA structure (Fig.1 B). To establish the function of these putative glycosylases, we first cloned the cDNAs for human NEIL1, NEIL2, and NEIL3, as well as the cDNA for mouse NEIL1 ortholog, by PCR. The NEIL3 protein appears to have an additional domain in the C-terminal half. This domain shows local homology to topoisomerase III (TOP3) and a C-terminal domain of APE2 (22Tsuchimoto D. Sakai Y. Sakumi K. Nishioka K. Sasaki M. Fujiwara T. Nakabeppu Y. Nucleic Acids Res. 2001; 29: 2349-2360Crossref PubMed Scopus (131) Google Scholar), a protein having an AP-endonuclease-like sequence with unknown function. We used in vitro expressed proteins to screen for enzymatic activity. As shown in Fig. 1 C, we could produce full-length NEIL1 and NEIL2 proteins using the rabbit reticulocyte lysate. For NEIL3, we were unable to obtain a full-size product and thus examined a truncated protein, NEIL3 (1–289), that is encoded by exons from 1 to 6. This polypeptide lacks the C-terminal domain but covers the entire Fpg-Nei-like domain. The translation mixtures were incubated with 5′-labeled 30-mer DNA containing a single Tg at a defined position to detect the strand incision activity. The reaction was conducted by inducing a strand incision at an abasic site to follow up a monofunctional DNA glycosylase having no incision activity. As shown in Fig.1 D, the rabbit lysate itself showed a slight background incision activity, probably because of rabbit enzymes (luciferase-expressing lane as a negative control in Fig.1 D). The reactions for NEIL2 and NEIL3 (1–289) did not yield nicked products above the background signal. On the other hand, the reactions for human NEIL1 and its mouse ortholog resulted in an obvious incision, suggesting that NEIL1 is a Tg-nicking DNA glycosylase. Because we anticipated that the NEIL1 gene product could be a back-up enzyme for Tg removal found in Nth1−/− mice, the mouse NEIL1 protein was characterized intensively. We produced a recombinant mouse NEIL1 tagged with His6 at its C terminus in E. coli (Fig.2 A, arrow) and recovered the protein from the soluble fraction of the whole cell extracts. Mouse NEIL1 was purified to apparent homogeneity by affinity chromatography through a Ni-NTA column and a HiTrap-SP ion-exchange column (Fig. 2 A) and analyzed for Tg-DNA nicking activity (Fig. 2 B). The yield (6 mg/liter culture) and the apparent homogeneity ensured that the observed Tg-DNA nicking activity (Fig.2 B) was derived from the recombinant NEIL1, rather than from contaminating host cell enzymes. This recombinant NEIL1 enzyme was used to further optimize the reaction conditions. The enzyme did not require a divalent cation, was resistant to EDTA up to 20 mm, and displayed comparable activity between pH 6 and 9 (data not shown). The activity was reduced markedly under high salt conditions (Fig.2 C). Because the specific activity decreased during storage (t 1/2 at −20 °C for ∼2 weeks), all quantitative experiments were performed within 2 days after purification of each batch. Purified recombinant human His6-NEIL1 protein exhibited similar Tg-DNA nicking activity (data not shown), indicating that both human and mouse NEIL1 are functional DNA glycosylases to Tg. By analogy with E. coli Fpg and Nei (23Tchou J. Kasai H. Shibutani S. Chung M.H. Laval J. Grollman A.P. Nishimura S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 4690-4694Crossref PubMed Scopus (693) Google Scholar, 24Jiang D. Hatahet Z. Melanmede R.J. Kow Y.W. Wallace S.S. J. Biol. Chem. 1997; 272: 32230-32239Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar), it can be expected that mammalian NEIL1 not only acts as DNA glycosylase but subsequently catalyzes β- and δ-elimination reactions that result in a strand nick with a one-nucleotide gap. We therefore examined the terminal structure of the 5′-incision and 3′-incision fragment at the position of the nick using 5′-labeled or 3′-labeled substrate, respectively. Analysis of the incision products in a high resolution gel revealed that the 5′-end of the 3′-incision fragment (Fig.3 A) or the 3′-end of the 5′-incision fragment (Fig. 3 B) had a phosphate group, which could be dephosphorylated by specific DNA phosphatases. From these data we concluded that the mammalian enzyme has (β-δ) AP lyase activity, as is the case with bacterial Fpg and Nei. Primary substrates for E. coli Nth and Nei are oxidized pyrimidines whereas Nei also shows 8-oxoguanine DNA glycosylase activity (25Blaisdell J.O. Hatahet Z. Wallace S.S. J. Bacteriol. 1999; 181: 6396-6402Crossref PubMed Google Scholar, 26Hazra T.K. Izumi T. Venkataraman R. Kow Y.W. Dizdaroglu M. Mitra S. J. Biol. Chem. 2000; 275: 27762-27767Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar). To determine the substrate specificity of mammalian NEIL1, we prepared oligonucleotide substrates containing a modified base at a defined position in the same sequence context. DNA lesions tested include two oxidized pyrimidine lesions, Tg and 5-OHU; an oxidized purine lesion, 8-oxoG; and an AP lesion. Using an amount of enzyme that cleaved about 50% of the Tg:A substrate as a reference, the nicking efficiency toward the other substrates was evaluated (Fig.4 A). NEIL1 cleaved Tg and 5-OHU to a similar extent but cleaved 8-oxoG much less efficiently. A further increase in the amount of NEIL1 added to 8-oxoG:C or 8-oxoG:A substrates invoked only marginal incision (Fig. 4 C). The nicking of the AP substrate was a little more efficient than that of the Tg:A substrate (Fig. 4 C). Interestingly, NEIL1 was able to cleave a single-strand substrate containing Tg, 5-OHU, and AP (Fig.4 A, lanes ss). Also, nicking at the modified base occurred with weak preference for the opposite DNA base (Fig.4 D, C ≅ T ≅ G ≥ A). NEIL1 (with an excess amount) did not cleave an unmodified base opposite Tg and 5-OHU (Fig.4 B), which implies that the enzyme does not produce a double-strand break at the lesion. Taken together, the findings indicate that the primary substrates of NEIL1 are oxidized pyrimidines in double-stranded, as well as single-stranded, DNA in any opposite base context. While examining the substrate specificity for NEIL1 and other NEIL proteins, we noticed that NEIL1 showed some activity toward uracil. As shown in Fig. 5, Aand B, the recombinant mouse NEIL1 protein incised the uracil-containing substrate only when the uracil was mispaired (U:C > U:G ≅ U:T). The incision activity of NEIL1 to U:C substrate was much stronger than that to 8-oxoG substrates (Fig. 4). Activity toward a U:A substrate could not be detected, whereas nicking of U:G and U:T was less efficient than that of U:C, and the plot of the nicking of U:G and U:T as a function of protein amount showed a threshold (Fig. 5, arrowheads). Because we showed that NEIL1 activity toward oxidized pyrimidines does not depend on the opposite base, we assumed that a major determinant for the observed nicking activity would be a mismatched state rather than the uracil base structure. Therefore, we also examined substrates containing the following simple pyrimidine mismatch: T:G, T:C, T:T (Fig.5 C), and C:A, C:C, C:T (Fig. 5 D). We observed that NEIL1 preferentially incised T:C, as well as U:C (Fig.5 C). The incisions toward the other mismatches occurred to some extent but again showed a threshold effect in the enzyme amount. Matched duplex DNA and single-stranded, unmodified DNA were not incised in these experiments (data not shown). Taken together, these results suggest that NEIL1 recognizes and nicks uracil and thymine mismatches with a preference of cytosine as the opposite base. Both mouse and human NEIL1 protein sequences show potential nuclear localization signals near the C terminus (see Fig.8). Indeed, as expected for a DNA repair protein, FLAG-tagged NEIL1 protein localized in the nucleus when expressed in COS-7 cells (Fig.6 A).Figure 6Subcellular localization and tissue expression. A, nuclear localization. NEIL1-FLAG was transiently expressed in COS-7 cells and detected by a FLAG and Alexa Fluor 488-conjugated second antibody. The nuclear DNA was visualized with 4′,6-diamidino-2-phenylindole (DAPI) staining.B, differential expressions of mouse Neil1 andNth1. An equalized cDNA panel (Clontech) was used for real-time, quantitative PCR with amplimers for Neil1, Nth1, andGapdh (control). The data were normalized by [Gapdh] and expressed relative to the level of [Nth1]/[Gapdh] in lung. He, heart; Br, brain;Sp, spleen; Lu, lung; Li, liver;SM, skeletal muscle; K i, kidney;Te, testis.View Large Image Figure ViewerDownload (PPT) It has been reported that mRNA for mouse NTH1 is not expressed homogeneously throughout the various tissues (27Sarker A.H. Ikeda S. Nakano H. Terato H. Ide H. Imai K. Akiyama K. Tsutui K., Bo, Z. Kubo K. Yamamoto K. Yasui A. Yoshida M.C. Seki S. J. Mol. Biol. 1998; 282: 761-774Crossref PubMed Scopus (74) Google Scholar). Because NTH1 and NEIL1 target the same Tg substrate, this redundancy might also be reflected at the level of tissue-dependent expression. To address whether Neil1 expression is ubiquitous or specific in tissues, we performed a quantitative PCR assay using an equalized tissue mouse cDNA panel. Fig. 6 B shows the expression levels of Nth1 and Neil1 compared in various tissues, relative to the mouse glyceraldehyde-3-phosphate dehydrogenase (Gapdh) used as a control. The highest expression levels of Nth1 are observed in oxygen-exposed lung, whereas the lowest Nth1 mRNA levels are detected in skeletal muscle (approximately 50 times lower than lung). For most tissues, expression of Neil1 is lower than that of Nth1. Exceptions are the spleen (comparable expression level toNeil1 and Nth1) and heart and skeletal muscle (Neil1 more abundantly" @default.
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