Title: Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA
Abstract: Article27 April 2006free access Repair deficient mice reveal mABH2 as the primary oxidative demethylase for repairing 1meA and 3meC lesions in DNA Jeanette Ringvoll Jeanette Ringvoll Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Line M Nordstrand Line M Nordstrand Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Cathrine B Vågbø Cathrine B Vågbø Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Vivi Talstad Vivi Talstad Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Karen Reite Karen Reite Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Per Arne Aas Per Arne Aas Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Knut H Lauritzen Knut H Lauritzen Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Nina Beate Liabakk Nina Beate Liabakk Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Alexandra Bjørk Alexandra Bjørk Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Richard William Doughty Richard William Doughty Department of Safety, PCS Biology, GE Healthcare Bio-sciences, Oslo, Norway Search for more papers by this author Pål Ø Falnes Pål Ø Falnes Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Department of Molecular Biosciences, University of Oslo, Oslo, Norway Search for more papers by this author Hans E Krokan Hans E Krokan Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Arne Klungland Corresponding Author Arne Klungland Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Department of Nutrition, Institute of Basic Medical Science, University of Oslo, Oslo, Norway Search for more papers by this author Jeanette Ringvoll Jeanette Ringvoll Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Line M Nordstrand Line M Nordstrand Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Cathrine B Vågbø Cathrine B Vågbø Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Vivi Talstad Vivi Talstad Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Karen Reite Karen Reite Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Per Arne Aas Per Arne Aas Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Knut H Lauritzen Knut H Lauritzen Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Nina Beate Liabakk Nina Beate Liabakk Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Alexandra Bjørk Alexandra Bjørk Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Search for more papers by this author Richard William Doughty Richard William Doughty Department of Safety, PCS Biology, GE Healthcare Bio-sciences, Oslo, Norway Search for more papers by this author Pål Ø Falnes Pål Ø Falnes Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Department of Molecular Biosciences, University of Oslo, Oslo, Norway Search for more papers by this author Hans E Krokan Hans E Krokan Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim Search for more papers by this author Arne Klungland Corresponding Author Arne Klungland Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway Department of Nutrition, Institute of Basic Medical Science, University of Oslo, Oslo, Norway Search for more papers by this author Author Information Jeanette Ringvoll1, Line M Nordstrand1, Cathrine B Vågbø2, Vivi Talstad2, Karen Reite1, Per Arne Aas2, Knut H Lauritzen1, Nina Beate Liabakk2, Alexandra Bjørk1, Richard William Doughty3, Pål Ø Falnes1,4, Hans E Krokan2 and Arne Klungland 1,5 1Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, Oslo, Norway 2Department of Cancer Research and Molecular Medicine, Norwegian University of Science and Technology, Trondheim 3Department of Safety, PCS Biology, GE Healthcare Bio-sciences, Oslo, Norway 4Department of Molecular Biosciences, University of Oslo, Oslo, Norway 5Department of Nutrition, Institute of Basic Medical Science, University of Oslo, Oslo, Norway *Corresponding author. Centre for Molecular Biology and Neuroscience and Institute of Medical Microbiology, Rikshospitalet-Radiumhospitalet HF, University of Oslo, 0027 Oslo, Norway. Tel.: +47 23074072; Fax: +47 23074061; E-mail: [email protected] The EMBO Journal (2006)25:2189-2198https://doi.org/10.1038/sj.emboj.7601109 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Two human homologs of the Escherichia coli AlkB protein, denoted hABH2 and hABH3, were recently shown to directly reverse 1-methyladenine (1meA) and 3-methylcytosine (3meC) damages in DNA. We demonstrate that mice lacking functional mABH2 or mABH3 genes, or both, are viable and without overt phenotypes. Neither were histopathological changes observed in the gene-targeted mice. However, in the absence of any exogenous exposure to methylating agents, mice lacking mABH2, but not mABH3 defective mice, accumulate significant levels of 1meA in the genome, suggesting the presence of a biologically relevant endogenous source of methylating agent. Furthermore, embryonal fibroblasts from mABH2-deficient mice are unable to remove methyl methane sulfate (MMS)-induced 1meA from genomic DNA and display increased cytotoxicity after MMS exposure. In agreement with these results, we found that in vitro repair of 1meA and 3meC in double-stranded DNA by nuclear extracts depended primarily, if not solely, on mABH2. Our data suggest that mABH2 and mABH3 have different roles in the defense against alkylating agents. Introduction Alkylated bases in DNA are repaired by three principally different mechanisms, depending on the type of lesion. These include (1) DNA glycosylases, which initiate base excision repair at methylated bases by removing the methylated base (Laval, 1977; Chen et al, 1989); (2) suicidal methyltransferases, which transfer the methyl group from the DNA onto a cysteine residue in the transferase itself (Tano et al, 1990) and (3) the newly identified AlkB family of dioxygenases, which directly reverse DNA base damage by oxidative demethylation (Duncan et al, 2002; Falnes et al, 2002; Trewick et al, 2002; Aas et al, 2003). The importance of efficient alkylation repair is further emphasized by the existence, within the same organism, of more than one enzyme representing each of these repair strategies. An example is the well-known AlkA and Tag DNA glycosylases, which initiate base excision repair at alkylated bases in Escherichia coli (E. coli) (Clarke et al, 1984). Tag is constitutively expressed, whereas AlkA is induced as part of the adaptive response (Karran et al, 1979; Evensen and Seeberg, 1982; Karran et al, 1982). All three strategies for alkylation repair are conserved from E. coli to man (Sedgwick, 2004). The conservation of enzymes specialized to repair methylated bases in DNA implies that naturally occurring methylating agents are widespread (Sedgwick, 2004). Such methylation can occur endogenously by the methyl donor S-adenosyl-methionine (SAM) (Barrows and Magee, 1982; Rydberg and Lindahl, 1982; Naslund et al, 1983) or by exposure to exogenous alkylating agents, originating from food, cigarette smoke, occupational chemicals or chemotherapeutics (Sedgwick, 2004). Methylating agents can react with DNA at several different sites on the DNA bases, including all the exocyclic oxygens and most ring nitrogens (Lindahl, 1993). The degree of alkylation at a given site depends not only on the mode of the chemical reaction (SN1 or SN2) but also on whether the DNA is in a single-stranded (ssDNA) or double-stranded (dsDNA) state (see below). AlkB has been thought to be involved in the cellular defense against methylation damage ever since it was discovered that alkB mutant E. coli cells were hypersensitive towards methylating agents (Kataoka et al, 1983; Kataoka and Sekiguchi, 1985), but it took almost 20 years to reveal the function of AlkB. A major breakthrough came when Sedgwick and co-workers demonstrated that AlkB repaired lesions that were specifically introduced in ssDNA, but not in dsDNA, and suggested that 1meA and 3meC were substrates for AlkB (Dinglay et al, 2000). Soon after, a landmark bioinformatics study placed the AlkB family of proteins in the superfamily of 2-oxoglutarate and Fe(II)-dependent oxygenases (Aravind and Koonin, 2001). By combining these clues, two independent studies showed that AlkB from E. coli was indeed a 2-oxoglutarate- and Fe(II)-dependent DNA repair enzyme capable of repairing 1meA and 3meC by oxidative demethylation (Falnes et al, 2002; Trewick et al, 2002). The substrate specificities of bacterial and human AlkB proteins have recently been extended to include 1-methylguanine (1meG) and 3-methylthymine (3meT), as well as ethylated bases (Delaney and Essigmann, 2004; Falnes, 2004; Koivisto et al, 2004). Thus, substrates for AlkB comprise all the lesions resulting from methylation at the N1 position of purines and N3 position of pyrimidines. These positions are structurally equivalent (the numbering of purine and pyrimidine bases is different) and shielded from methylation in the context of dsDNA, as they are involved in hydrogen bonding in Watson–Crick base pairing. Recently, the substrates of AlkB was expanded to include 1,N(6)-ethenoadenine and 3,N(4)-ethenocytosine (Delaney et al, 2005). Bioinformatics indicate the existence of eight different dioxygenases of the AlkB family in mammalian cells, denoted ABH1–ABH8 (AlkB Homolog) (Kurowski et al, 2003). However, only two of the corresponding human proteins, hABH2 and hABH3, have been shown to possess a similar repair activity as AlkB from E. coli (Duncan et al, 2002; Aas et al, 2003). Besides removing lesions from DNA, AlkB and hABH3 also repair 1meA and 3meC in RNA (Aas et al, 2003). Purified AlkB and hABH3 were recently found to restore the biological function of mRNA and tRNA inactivated by chemical methylation (Ougland et al, 2004). Furthermore, chemically methylated E. coli tRNA was demethylated in an AlkB-overexpression strain, revealing a putative role for such repair enzymes in vivo (Ougland et al, 2004). In contrast, purified hABH2 displayed undetectable or very low levels of activity against methylated RNA species (Aas et al, 2003; Ougland et al, 2004). Whereas ds nucleic acids are the favored substrates for hABH2, ss substrates are preferred by hABH3 (Falnes et al, 2004; Koivisto et al, 2004), and these proteins also display distinct subnuclear localization patterns (Aas et al, 2003). An elegant study by Delaney and Essigmann (2004) investigated the relative mutagenicity, genotoxicity and repair of 1meA, 3-ethylcytosine (3eC), 1meG and 3meT in E. coli. All these lesions blocked replication in repair-deficient cells, whereas their miscoding properties varied significantly. Mutations observed included G to T and G to A base substitutions, which are also observed in organisms exposed to alkylating agents (Delaney and Essigmann, 2004). It was concluded that AlkB suppresses both genotoxicity and mutagenicity at physiologically realistic levels of alkylated lesions. In order to investigate the relevance of DNA repair by ABH2 and ABH3 in vivo, we have generated gene-targeted mice lacking functional genes coding for these two proteins. Novel assays have been developed to measure the presence of methyl lesions in mouse genomic DNA, as well as to characterize the repair proficiency of extracts and cells prepared from mouse tissues. Results Generation of mABH2 and mABH3 null mice The two murine AlkB homologs, mAHB2 and mABH3, are relatively small basic proteins spanning 239 and 286 amino acids, respectively. The identity between mABH2 and hABH2 is 75.1% and the identity between mABH3 and hABH3 is 85.7%, suggesting similar activities (Lee et al, 2005). Sequence alignment shows that the three residues (His 149/191, Asp 151/193 and His 214/257), presumed to constitute the Fe(II)-binding cluster in E. coli AlkB, are conserved in the mouse proteins. The importance of some of these residues was recently verified by site-directed mutagenesis of mouse and human ABH2 and ABH3 (Lee et al, 2005). Based on comparison with other 2-oxoglutarate and Fe(II)-dependent oxygenases, a conserved arginine (Arg 226 in mABH2 and Arg 269 in mABH3) most likely binds the 5-carboxylate of the 2-oxoglutarate co-substrate (Mukherji et al, 2001; Aas et al, 2003; Valegard et al, 2004). The Fe(II)-binding cluster and the conserved arginine are encoded by exon 4 of the mABH2 gene and exons 8 and 10 of the mABH3 gene. To eliminate activity of the enzymes, exon 4 of mABH2 and exons 7 and 8 of mABH3 were targeted, as outlined in Figures 1 and 2 and Supplementary Figure 1. Figure 1.Targeted disruption of the murine mABH2 locus. (A) Overview of the mABH2 exons with the location of the 2-oxoglutarate- and Fe(II)-dependent oxygenase domain as well as the AlkB domain. Translated regions are shown in red, whereas untranslated regions are in blue. (B) Physical map of the genomic DNA containing the mABH2 gene with the location of exons 1, 2, 3 and 4. Genomic fragments were subcloned on both sides of the neo gene to generate a construct with a deleted 3.2 kb fragment, including exon 4. The positions of the 5′ and 3′ probes used to screen ES cells for correct targeting events are indicated, as are the restriction enzymes relevant for subcloning and genotyping by Southern hybridization. (C) Correct 5′ and 3′ targeting, left and right panel, of mABH2 mice were analyzed by PCR. Details of ES-cell genotyping are outlined in Supplementary Figure 1. Download figure Download PowerPoint Figure 2.Targeted disruption of the murine mABH3 locus. (A) Overview of the mABH3 exons, as for mABH2 above. (B) Physical map of the genomic DNA containing parts of the mABH3 gene with the location of exons 4, 5, 6, 7 and 8. Genomic fragments were subcloned on both sides of the neo gene to generate a construct with a deleted 5.6 kb fragment including exon 8 and part of exon 7. The position of the 3′ probe is shown on the illustration for the genomic DNA, whereas the positions of the primers (Supplementary Table 1) used for PCR screening of ES-cells are indicated on the targeted allele. Restriction enzymes relevant for subcloning and genotyping by Southern analysis are indicated. (C) Correct 5′ and 3′ targeting, left and right panel, of mABH3 mice were analyzed by PCR. Details of ES-cell genotyping are outlined in Supplementary Figure 1. Other details as in Figure 1. Download figure Download PowerPoint Mendelian inheritance of mutated alleles in single and doubleknockouts Genotyping of more than 40 live-born mice produced by inter-mating of F1 heterozygotes (one litter for each genotype is presented in Supplementary Figure 1F) showed that mABH2 and mABH3 null mice are viable. Homozygous mutant mice were recovered in F2 litters at frequencies consistent with Mendelian segregation (data not shown) and were phenotypically indistinguishable from their heterozygous and wild-type siblings. Furthermore, interbreeding of mABH2 and mABH3 null mice generated homozygous double mutants at the expected ratio in F2. Null animals remained viable and apparently healthy into adulthood with no overt phenotype. Although some mABH2 and mABH3 null mice have died before the age of 18 months, no obvious phenotype was observed (results not shown). The aging colonies of mice are now about 18-month old, except for the double knockouts at 12 months of age. Histopathological examination of four 18-month-old mice, each of wild-type, mABH2 and mABH3, and two 12-month-old double knockouts, revealed no lesions or abnormalities that were considered to be related to genotype (Materials and methods). All lesions recorded were considered to be incidental. Accumulation of 1meA in repair-deficient mice To study the role of mABH2 and mABH3 for repair of erroneous endogenous methylation in DNA, genomic DNA was extracted from the livers of untreated wild-type, mABH2 and mABH3 knockout mice at different ages (Figure 3). The steady-state level of 1meA was determined at 1, 4, 8 and 12 months of age by the HPLC-MS/MS scheme, described in detail in Materials and methods. Two DNA samples, typically 100 μg, were purified from each liver, and 2–4 mice of each genotype were sacrificed at each time point. The 1meA levels were below the detection limit in untreated 1-month-old mice, regardless of the genotype. Likewise, no 1meA could be detected in wild-type and mABH3 knockout mice at later time points. In contrast, 1meA accumulation was observed in 4-month-old mABH2 null mice, although only slightly above the detection limit, with a further increase at 8 months (Figure 3A and B). We estimated that at 8 months of age, wild-type and mABH3 knockout mice had accumulated less than 120 1meA per genome, whereas the corresponding number for the mABH2 knockout was within the range 150–500. Significant accumulation of 1meA was also observed in two 12-month-old mABH2 knockout mice. These results suggest that a substantial degree of aberrant methylation, producing 1meA lesions in DNA, occurs under normal physiological conditions. Figure 3.Accumulation of 1meA in aging repair-deficient mice. (A) Scatter diagram showing the accumulation of 1meA in genomic DNA from liver of 1-, 4-, 8- and 12-month-old wild-type, mABH2-and mABH3-targeted mice. Only 8- and 12-month-old mABH2-targeted mice had 1me(dA)-levels above the detection limit, which was approximately 3.5 1me(dA):108dA. Duplicates of 100 μg genomic DNA from two to four mice of each genotype were used at each time point. The steady-state level of 1meA was determined by the HPLC-MS/MS scheme described in the Materials and methods. (B) Numerical values of 1meA in genomic DNA from liver of 8- and 12–month-old mABH2-targeted mice plotted in panel A, with parallel determinations denoted as a and b. (C) HPLC-MS/MS chromatogram of 1me(dA) in genomic DNA from liver of 8-month-old wild-type (upper panel) and mABH2-targeted mice (middle panel), compared with 1me(dA) standard (lower panel). Download figure Download PowerPoint Kinetics of 1meA repair in vivo To assess the role of 1meA repair in living cells, mouse embryo fibroblast (MEF) cell lines established from the different mouse models were treated with the DNA methylating agent methyl methane sulfonate (MMS). Before examining the repair capacities in targeted cell lines, the repair kinetics of 1meA lesions induced by three different MMS concentrations (0.1, 0.5 and 2 mM) in wild-type cells were established (Figure 4A), and a substantial degree of repair was observed upon exposure to all three concentrations. The highest concentration of MMS (2 mM) was subsequently used in all the studies owing to more robust and accurate lesion detection. Repair was allowed to continue for up to 36 h after removal of MMS. Genomic DNA was purified, and the amount of 1meA determined using HPLC-MS/MS (Figure 4). Extensive washing of the cells was essential to ensure that no residual MMS remained as this caused a substantial further increase of DNA methylation (results not shown). In wild-type cells exposed to 2 mM MMS for 1 h, the amount of 1meA present was reduced by more than 50% after 8 h; approximately 10% of the induced 1meA remained in the cells after 36 h (Figure 4A and B). Such repair kinetics are comparable to those observed for other base lesions (Klungland et al, 1999). Figure 4.Kinetics of 1meA repair in vivo. (A) Kinetics of 1meA repair in wild-type MEF cells treated with 0.1, 0.5 and 2 mM of MMS. Cells were incubated with MMS for 1 h at 37°C, washed extensively and then allowed to repair DNA damages for up to 36 h before DNA isolation and HPLC-MS/MS identification of 1meA. One point represents approximately 106 cells and 50 μg of genomic DNA. (B) Kinetics of 1meA removal in wild-type, mABH2- and mABH3-targeted cells treated with 2 mM MMS. Further details are the same as for panel A. (C) Repair kinetics in replication arrested wild-type and mABH2-depleted cells incubated with 6 μM aphidicolin. Cells were treated with 2 mM of MMS for 1 h at 37°C, and then incubated with 6 μM aphidicolin for up to 36 h to investigate the DNA repair process under replication arrest. DNA isolation and HPLC-MS/MS identification of 1meA were carried out as described for panel B above. Download figure Download PowerPoint The repair kinetics of 1meA in mABH3-targeted cells was indistinguishable from those of wild-type cells (Figure 4B). In contrast, and in agreement with the data obtained regarding repair of 1meA and 3meC in cellular extracts presented below, only a small fraction of 1meA was repaired in mABH2-targeted cells. An approximate 10% reduction in the 1meA levels was observed after 8 h, whereas a reduction of 30% was observed after 36 h. All experiments were carried out in MEF cells grown to about 80% confluence. In order to eliminate dilution of the lesions due to DNA replication, the experiments were repeated in the mABH2-targeted cells with the addition of aphidicolin, which inhibits elongation of the replicons (Saintigny et al, 2001). The repair rate of 1meA in mABH2-targeted cells was further reduced with this treatment, and only a negligible fraction of 1meA was removed following 36 h incubation (Figure 4C). In order to exclude the possibility that aphidicolin inhibits 1meA repair, similar experiments were carried out in wild-type cells. No significant reduction in the repair rates was observed (Figure 4C). Cellular sensitivity to methylating agent E. coli mutants deficient in 3-methyladenine DNA glycosylase activity (alkA and alkA tag) are extremely sensitive to exposure of alkylating agents (Clarke et al, 1984), whereas the alkB mutant displays moderate sensitivity (Kataoka et al, 1983). MEFs and ES clones lacking the murine 3-methyladenine DNA glycosylase (Aag) are hypersensitive towards methylating agents (Engelward et al, 1996; Engelward et al, 1997), but much less so than in the E. coli alkA tag mutant. Based on these observations, a modest but significant sensitizing of mABH2 cells and wild-type resistance of mABH3 cells was expected (Figure 5). Wild-type growth rates are observed in all cell lines. Figure 5.Effect of MMS on growth of MEF cell lines. Cells were seeded at a density of 2000 cells per well in 96-well plates (n=16 for each measurement). Wild-type cell lines (Wt), mABH2 or mABH3 cell lines were used, as indicated. After 24 h culturing, the cells were treated with MMS continuously (A) or for 1 h (B). The results were expressed as cell numbers in MMS-treated cultures compared to untreated controls. (A) Cells treated for 48 or 72 h in media containing 0–500 μM MMS. (B) Cells were treated for 1 h with media containing 0–5 mM MMS, then washed with PBS before 200 μl fresh medium was added. Cell numbers were measured using the MTT-assay. Untreated MEF cell lines of the three genotypes had essentially similar growth rates. Download figure Download PowerPoint DNA substrates for 1meA and 3meC repair Purified E. coli AlkB and the human homologs hABH2 and hABH3 are able to repair 1meA and 3meC in DNA by oxidative demethylation (Duncan et al, 2002; Falnes et al, 2002; Trewick et al, 2002; Aas et al, 2003). Taking advantage of the inability of the DpnII restriction endonuclease to recognize a methylated GATC sequence, we established an in vitro assay for the repair of 1meA and 3meC by cellular extracts (Figure 6A and Supplementary Table 1). Repair of these lesions was monitored using a ds oligonucleotide containing either 1meA or 3meC within the GATC recognition sequence, and DpnII cleavage would only occur if the lesion had been repaired by direct reversal. Initially, the BamHI site was used to distinguish between methylated and unmethylated GGATCC. However, some BamHI activity was observed on methylated sequences (results not shown). As illustrated in Figure 6B, 49-mer 5′-[32P]end-labeled oligonucleotides containing 1meA or 3meC were cleaved to 22 nucleotide (nt)-labeled fragments only when the DpnII digestion was preceded by incubation with AlkB, hABH2 or hABH3. A similar approach, utilizing methylation-sensitive restriction endonucleases, has previously been used successfully for the characterization of O6-alkylguanine-DNA alkyltransferase activity (Wu et al, 1987). Figure 6.DNA substrates for 1meA and 3meC repair and activity of AlkB, hABH2 and hABH3. (A) The 1meA substrate consists of a 49 bp oligonucleotide with a methylated adenine in position 24, radioactively labeled at the 5′-end and hybridized to a complementary strand. The 3meC substrate has a methylated cytosine in position 26. The methylated base is located in the recognition site of the restriction enzyme DpnII. To visualize the repair, we used the enzyme DpnII, which is methylation sensitive and only cleaves the substrate if the methyl group is removed. An unrepaired substrate will result in a band of 49 nt, whereas the repaired and cleaved substrate will appear as a 22 nt band following denaturing PAGE. (B) Activity of purified AlkB, hABH2 and hABH3 on 1meA and 3meC in a dsDNA oligonucleotide. DNA substrates were incubated with purified enzymes as indicated, digested with DpnII and separated by 20% denaturing PAGE. Labeled DNA was visualized by phosphorimaging. Both substrates were incubated with DpnII, without previous repair, as a negative control. Download figure Download PowerPoint Activity of hABH2 on dsDNA has been reported to be stimulated by magnesium, and maximal repair activity for hABH2 was obtained in the presence of 10 mM MgCl2 (Falnes et al, 2004). In contrast, AlkB and hABH3 were not affected by magnesium. Consequently, we added magnesium to the reaction buffer in the case of mABH2, but not for AlkB or mABH3. Figure 6B demonstrates that hABH3 repairs 3meC more efficiently than 1meA. This is consistent with previous reports (Duncan et al, 2002; Aas et al, 2003; Lee et al, 2005). The repair of synthetic oligonucleotides by purified AlkB family enzymes was carried out to justify further use of these DNA substrates, and not to reveal the kinetics of the purified enzymes. Enzyme activity in extracts from mABH2, mABH3 and mABH2/mABH3 null mice Human ABH2 and ABH3 have previously been characterized in vitro. Purified hABH2 and hABH3 repair both 1meA and 3meC in DNA, although with a different preference for ds versus ss substrates (see Introduction). In order to assess the properties and relevance of these repair enzymes in cellular extracts, the duplex oligonucleotides described above were incubated with nuclear extracts from liver, testis and kidney, cleaved with DpnII, and the reaction products analyzed by denaturing PAGE. MgCl2 (10 mM) was added for optimal activity of mABH2. In addition, the amount of extract, DNA substrate and buffer conditions were titrated to obtain maximum repair activity and to minimize degradation of the DNA substrate (results not shown). The results indicate that mABH2 is the primary activity for repair of 1meA and 3meC in dsDNA in nuclear extracts (Figure 7 and Supplementary Figure 3).