Repair of Methylation Damage in DNA and RNA by Mammalian AlkB Homologues
2005; Elsevier BV; Volume: 280; Issue: 47 Linguagem: Inglês
10.1074/jbc.m509881200
ISSN1083-351X
AutoresDong-Hyun Lee, Seung‐Gi Jin, Sheng Cai, Yuan Chen, Gerd P. Pfeifer, Timothy O’Connor,
Tópico(s)RNA Research and Splicing
ResumoHuman and Escherichia coli derivatives of AlkB enzymes remove methyl groups from 1-methyladenine and 3-methylcytosine in nucleic acids via an oxidative mechanism that releases the methyl group as formaldehyde. In this report, we demonstrate that the mouse homologues of the α-ketoglutarate Fe(II) oxygen-dependent enzymes mAbh2 and Abh3 have activities comparable to those of their human counterparts. The mAbh2 and mAbh3 release modified bases from both DNA and RNA. Comparison of the activities of the homogenous ABH2 and ABH3 enzymes demonstrate that these activities are shared by both sets of enzymes. An assay for the detection of α-ketoglutarate Fe(II) dioxygenase activity using an oligodeoxyribonucleotide with a unique modification shows activity for all four enzymes studied and a loss of activity for eight mutant proteins. Steady-state kinetics for removal of methyl groups from DNA substrates indicates that the reactions of the proteins are close to the diffusion limit. Moreover, mAbh2 or mAbh3 activity increases survival in a strain defective in alkB. The mRNAs of AHB2 and ABH3 are expressed most in testis for ABH2 and ABH3, whereas expression of the homologous mouse genes is different. The mAbh3 is strongly expressed in testis, whereas highest expression of mAbh2 is in heart. Other purified human AlkB homologue proteins ABH4, ABH6, and ABH7 do not manifest activity. The demonstration of mAbh2 and mAbh3 activities and their distributions provide data on these mammalian homologues of AlkB that can be used in animal studies. Human and Escherichia coli derivatives of AlkB enzymes remove methyl groups from 1-methyladenine and 3-methylcytosine in nucleic acids via an oxidative mechanism that releases the methyl group as formaldehyde. In this report, we demonstrate that the mouse homologues of the α-ketoglutarate Fe(II) oxygen-dependent enzymes mAbh2 and Abh3 have activities comparable to those of their human counterparts. The mAbh2 and mAbh3 release modified bases from both DNA and RNA. Comparison of the activities of the homogenous ABH2 and ABH3 enzymes demonstrate that these activities are shared by both sets of enzymes. An assay for the detection of α-ketoglutarate Fe(II) dioxygenase activity using an oligodeoxyribonucleotide with a unique modification shows activity for all four enzymes studied and a loss of activity for eight mutant proteins. Steady-state kinetics for removal of methyl groups from DNA substrates indicates that the reactions of the proteins are close to the diffusion limit. Moreover, mAbh2 or mAbh3 activity increases survival in a strain defective in alkB. The mRNAs of AHB2 and ABH3 are expressed most in testis for ABH2 and ABH3, whereas expression of the homologous mouse genes is different. The mAbh3 is strongly expressed in testis, whereas highest expression of mAbh2 is in heart. Other purified human AlkB homologue proteins ABH4, ABH6, and ABH7 do not manifest activity. The demonstration of mAbh2 and mAbh3 activities and their distributions provide data on these mammalian homologues of AlkB that can be used in animal studies. Environmental and endogenous agents constantly subject DNA and RNA to damage. Alkylating agents cover a spectrum of chemicals that in their most simple form methylate DNA or RNA. The subsequent distribution of methylated products of the reaction of methylating agents with nucleic acids depends on the nature of the agent. SN1 alkylating agents react via a carbonium ion intermediate whereas SN2 agents interact directly with DNA (1Singer B. Grunberger D. Molecular Biology of Mutagens and Carcinogens. Plenum, New York1983Crossref Google Scholar). Under these circumstances different sites on nucleic acids are modified. In addition to the alkylating agent, the structure of the DNA also plays a role. Regions of single-stranded nucleic acids favor reaction at sites that are virtually untouched in double-stranded nucleic acids. For example, position 1 of Ade is not modified to any extent in double-stranded DNA (ds-DNA), 2The abbreviations used are:dsdouble-stranded2OGα-ketoglutarate Fe(II)-dependent dioxygenaseABHhuman AlkB homologuemAbhmouse AlkB homologueODNoligodeoxyribonucleotidepoly(A)polyribonucleotide Apoly(C)polyribonucleotide Cpoly(dA)polydeoxyribonucleotide Apoly(dC)polydeoxyribonucleotide Csssingle-strandedNTAnitrilotriacetic acid[3H]MNUN-[3H]methyl-N-nitrosoureaDMSdimethylsulfate whereas in single-stranded DNA (ss-DNA) it becomes a major target (1Singer B. Grunberger D. Molecular Biology of Mutagens and Carcinogens. Plenum, New York1983Crossref Google Scholar, 2Margison G.P. O'Connor P.J. Grover P.L. Chemical Carcinogenesis and DNA. Vol. I. CRC Press, 1979Google Scholar, 3Shooter K.V. Slade T.A. Chem. Biol. Interact. 1977; 19: 353-361Crossref PubMed Scopus (42) Google Scholar, 4Lawley P.D. Shooter K.V. House W.L. Shah S.A. Biochem. J. 1971; 122: 22PCrossref PubMed Google Scholar). double-stranded α-ketoglutarate Fe(II)-dependent dioxygenase human AlkB homologue mouse AlkB homologue oligodeoxyribonucleotide polyribonucleotide A polyribonucleotide C polydeoxyribonucleotide A polydeoxyribonucleotide C single-stranded nitrilotriacetic acid N-[3H]methyl-N-nitrosourea dimethylsulfate In 1983, an Escherichia coli mutant with sensitivity to SN2 alkylating agents was isolated and named alkB (5Kataoka H. Yamamoto Y. Sekiguchi M. J. Bacteriol. 1983; 153: 1301-1307Crossref PubMed Google Scholar). The cloning of the gene and the acknowledgment that it was linked to the ada operon responding to DNA alkylation damage produced enormous interest in this gene (6Kondo H. Nakabeppu Y. Kataoka H. Kuhara S. Kawabata S. Sekiguchi M. J. Biol. Chem. 1986; 261: 15772-15777Abstract Full Text PDF PubMed Google Scholar, 7Kataoka H. Sekiguchi M. Mol. Gen. Genet. 1985; 198: 263-269Crossref PubMed Scopus (50) Google Scholar). Despite the knowledge that expression of alkB in human cells could increase resistance to alkylating agents (8Samson L. Derfler B. Waldstein E.A. Proc. Natl. Acad. Sci. U. S. A. 1986; 83: 5607-5610Crossref PubMed Scopus (126) Google Scholar, 9Chen B.J. Carroll P. Samson L. J. Bacteriol. 1994; 176: 6255-6261Crossref PubMed Google Scholar), AlkB had no known enzymatic function. The genetic work in E. coli was important, but without function, progress in the line of AlkB lagged behind that of other DNA repair studies (10Volkert M.R. Environ. Mol. Mutagen. 1988; 11: 241-255Crossref PubMed Scopus (95) Google Scholar, 11Volkert M.R. Gately F.H. Hajec L.I. Mutat. Res. 1989; 217: 109-115Crossref PubMed Scopus (30) Google Scholar, 12Volkert M.R. Hajec L.I. Nguyen D.C. J. Bacteriol. 1989; 171: 1196-1198Crossref PubMed Google Scholar). Even the isolation of a human homologue of AlkB, ABH1, failed to provide insight as to function of this group of proteins (13Wei Y.F. Carter K.C. Wang R.P. Shell B.K. Nucleic. Acids Res. 1996; 24: 931-937Crossref PubMed Scopus (60) Google Scholar). Using a host cell reactivation assay, alkB mutants were shown to efficiently process chemically methylated ds-DNA phage, but not chemically methylated ss-DNA phage (14Dinglay S. Gold B. Sedgwick B. Mutat. Res. 1998; 407: 109-116Crossref PubMed Scopus (34) Google Scholar, 15Dinglay S. Trewick S.C. Lindahl T. Sedgwick B. Genes. Dev. 2000; 14: 2097-2105PubMed Google Scholar). That provided an important clue to the nature of AlkB. Crucial information in elucidating the enzymatic function of AlkB was provided by bioinformatics analysis that showed AlkB belonged to a large class of enzymes known as 2-oxoglutarate- and iron-dependent dioxygenases (16Aravind L. Koonin E.V. Genome. Biol. 2001; 2: 7.1-7.8Crossref Google Scholar). Once the link between the AlkB and the 2OG family was established, two groups showed that AlkB was in fact a member of the family and that AlkB released methyl groups not only from 1-meAde and 3-meCyt, but also 3-meThy and 1-meGua (17Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (634) Google Scholar, 18Falnes P.O. Johansen R.F. Seeberg E. Nature. 2002; 419: 178-182Crossref PubMed Scopus (504) Google Scholar, 19Koivisto P. Robins P. Lindahl T. Sedgwick B. J. Biol. Chem. 2004; 279: 40470-40474Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 20Falnes P.O. Bjoras M. Aas P.A. Sundheim O. Seeberg E. Nucleic Acids Res. 2004; 32: 3456-3461Crossref PubMed Scopus (99) Google Scholar, 21Delaney J.C. Essigmann J.M. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 14051-14056Crossref PubMed Scopus (202) Google Scholar). The identification of an activity for AlkB accelerated the field, and two homologues of human origin, ABH2 and ABH3, were isolated based on BLAST searches of databases. These proteins were both demonstrated to be active on methylated substrates (22Aas P.A. Otterlei M. Falnes P.O. Vagbo C.B. Skorpen F. Akbari M. Sundheim O. Bjoras M. Slupphaug G. Seeberg E. Krokan H.E. Nature. 2003; 421: 859-863Crossref PubMed Scopus (521) Google Scholar, 23Duncan T. Trewick S.C. Koivisto P. Bates P.A. Lindahl T. Sedgwick B. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 16660-16665Crossref PubMed Scopus (314) Google Scholar) (Fig. 1, a and b). Although ABH2 and ABH3 were active, ABH1, the first member in the ABH series, was not active in biochemical or complementation assays. Another bioinformatics search revealed a number of potential members of the series (24Kurowski M.A. Bhagwat A.S. Papaj G. Bujnicki J.M. BMC Genomics. 2003; 4: 48Crossref PubMed Scopus (165) Google Scholar). This suggests that there are a number of homologues related to AlkB that cover most phyla. One method to elucidate biological and biochemical function is to work in related organisms. Probably the most studied mammal aside from humans is mouse. The model presented by mouse has been exploited by targeted deletion of genes by homologous recombination. Therefore, we have isolated cDNAs coding for two mouse homologues of ABH2 and ABH3. We have expressed those sequences, produced proteins that were purified to homogeneity, demonstrated activity on both DNA and RNA substrates, determine the kinetics for the release of methyl groups from DNA, and show the tissue distribution in mouse and human of the RNA for both genes. We also have isolated and expressed cDNAs for ABH4-8 and discuss the results of experiments on the purported relation between these proteins and the activities of ABH2 and ABH3. Strains—The F′-plasmid was introduced into E. coli strains HK81 (wild type) and HK82 (alkB) as described (15Dinglay S. Trewick S.C. Lindahl T. Sedgwick B. Genes. Dev. 2000; 14: 2097-2105PubMed Google Scholar) to generate the strains HK81/F′ and HK82/F′, respectively (the HK81 and HK82 strains were kindly provided by Michael Volkert) (5Kataoka H. Yamamoto Y. Sekiguchi M. J. Bacteriol. 1983; 153: 1301-1307Crossref PubMed Google Scholar, 7Kataoka H. Sekiguchi M. Mol. Gen. Genet. 1985; 198: 263-269Crossref PubMed Scopus (50) Google Scholar, 11Volkert M.R. Gately F.H. Hajec L.I. Mutat. Res. 1989; 217: 109-115Crossref PubMed Scopus (30) Google Scholar). Briefly, overnight cultures of XL1-Blue (donor), HK81 (recipient), and HK82 (recipient) were diluted 20-fold and grown to approximately A600 0.5. Cells were mixed in ratio donor/recipient = 1:10 and incubated at 37 °C for 30 min. The cell suspension was streaked out on LB plates with 50 μg/ml streptomycin and 10 μg/ml tetracycline. HK82 (alkB DE3) was constructed using the λDE3 lysogenization kit (Novagen) according to the manufacturer's instructions. The expression and purification of all the human or mouse AlkB homologues were conducted in this strain. Purification of Human ABH2 and ABH3 and Mouse mAbh2 and mAbh3—Human ABH2 and ABH3 cDNAs were obtained by RT-PCR with total RNA of LNCaP prostate cancer cell line. Mouse Abh2 and Abh3 cDNAs were obtained from the IMAGE consortium, and IMAGE Clone IDs for expressed sequence tags (EST) clones are 4196765 (mABH2) and 3993214 (mABH3). The cDNAs were amplified by PCR using Pfu Turbo DNA polymerase, and subcloned into the EcoRI/XhoI sites (ABH2) or BamHI/SalI sites (ABH3, mABH2, and mABH3) of the expression vector pET28a(+) (Novagen). All sequences for mAbh2, mAbh3, ABH4-ABH8 have been submitted to GenBank™. His-tagged proteins were expressed in E. coli strain HK82 (alkB DE3) in LB medium. Bacterial pellets were lysed and sonicated in ice-cold lysis buffer (50 mm NaH2PO4, 300 mm NaCl, 10 mm imidazole) with 1 mg/ml of lysozyme. The lysate was centrifuged at 10,000 × g for 25 min at 4 °C, and the extracts were loaded onto a Ni-NTA flow column, which was washed first with lysis buffer containing 20 mm imidazole. The bound proteins were released from the column with elution buffer (50 mm NaH2PO4, 300 mm NaCl, and each of 50 mm, 100 mm and 250 mm imidazole, respectively) (Qiagen). We further purified the fractions obtained from the Ni-NTA columns by ion exchange with CM Sepharose® Fast Flow and FPLC with Superose® 12 HR 10/30 column (Amersham Biosciences). Fractions were analyzed by SDS-PAGE, and the relevant fractions were pooled. Point mutations in human or mouse AlkB homologues were introduced by QuikChange® II XL site-directed mutagenesis kit (Stratagene) according to the manufacturer's instructions. The sequences of the mutant human ABH2 (D173A), ABH2 (H236A), human ABH3 (D193A), human ABH3 (H257A), mAbh2 (D151A), mAbh2 (H214A) mAbh3, and (D193A), mAbh3 (H257A) were confirmed by sequencing the entire coding sequence. The mutant proteins were expressed and purified in the same manner as the wild-type proteins. Preparation of Methylated DNA and RNA—Poly(A), poly(C), poly(dA), and poly(dC) were obtained from Amersham Biosciences. The homopolymers, single-stranded M13mp18, and single-stranded DNA oligo 5′-AAAGCAAGAAACGAAAAAGCGAAA-3′ (40 μg) were incubated in sodium cacodylate buffer (50 mm, pH 7.25) in the presence of 0.1 mCi of N-[3H]methyl-N-nitrosourea (Amersham Biosciences) at 37 °C for 2 h. The nucleic acids were precipitated by the addition of NaCl (final concentration of 1 m) and 2.5 volume of 100% ethanol. The ethanol precipitation step was repeated once more, followed by dialysis against 10 mm Tris buffer (pH 8.0) to remove traces of free radioactivity. For generation of double-stranded 3H-methylated substrate, the single-stranded [3H]CH3-ODN was incubated in the presence of a 3-fold molar excess of non-methylated complementary strand for 20 min at 37 °C in 10 mm Tris buffer (pH 8.0) and then placed on ice. The double-stranded substrate was generated immediately prior to the experiment. The ss-M13mp18 DNA was treated with various concentrations of MMS in 100 mm sodium cacodylate (pH 7.25) for 30 min at 30 °C, followed by ethanol precipitation of the DNA, and resuspension in 10 mm Tris buffer (pH 8.0). The ss-DNA ODNs with one 1-meA or 3-meC (ChemGenes) were obtained from the Midland Certified Reagent Company. Assay for Complementation Using MMS-treated M13mp18 ss—DNA-competent cells (HK81/F′ and HK82/F′) were prepared by treatment with cold 0.1 m CaCl2 immediately prior to the experiment. Incubation of MMS-treated M13 ss-DNA (1 μg) in the absence or presence of mammalian AlkB homologues (10 pmol) was performed in reaction mixture, containing 50 mm Hepes-KOH (pH 8), 40 μm FeSO4, 1 mm 2-oxoglutarate, 2 mm ascorbate, 50 μg/ml bovine serum albumin for 1 h. After the reaction, the ss-DNA was recovered by ethanol precipitation, and 200 ng was used for each transformation. Release of Radioactivity by 2OG Activity of AlkB Homologues—[3H]MNU-treated DNA or RNA (typically 0.2 μg, containing 1000 dpm. label) was incubated in reaction mixture for 30 min at 37 °C in the presence of varying amounts of mammalian AlkB homologues in the following buffer: 50 mm Hepes-KOH (pH 8), 40 μm FeSO4, 1 mm 2-oxoglutarate, 2 mm ascorbate, 50 μg/ml bovine serum albumin. Subsequently, nucleic acids were ethanol-precipitated in the presence of carrier calf thymus DNA, and the radioactivity in the supernatant was measured by scintillation counting. Assay for Repair of 1-meA or 3-meC in Site Specifically Modified ODNs—The sequences of ss-DNA oligos with a 1-methyl dA or 3-methyl dC were as follows: 5′-AAAGCAGmAATTCGAAAAAGCGAAA-3′, 5′-AAAGCACmCGGTCGAAAAAGCGAAA-3′. The ss-DNA oligos (at least in 3-fold molar excess compared with the ABH2 or 3) were labeled with [γ-32P]ATP at the 5′-end, and incubated with 50 pmol of human or mouse ABH2 and ABH3 and for 30 min at 37 °C in the same buffer as for the complementation assay. After phenol/chloroform extraction and ethanol precipitation, ss-DNA oligos were annealed with a 3-fold molar excess of non-methylated complementary strand for 20 min at 37 °C in 10 mm Tris buffer (pH 8.0), and then subjected to EcoRI or HpaII treatment for 30 min at 37 °C, followed by phenol/chloroform extraction and ethanol precipitation. The restriction enzyme cleavage was verified on 20%(w/v) polyacrylamide gels containing 7 m urea. The gels were dried and exposed overnight to storage phosphor screens and were scanned at 200 micron resolution using a Typhoon 9410 Variable using ImageQuant 5.2 software (Molecular Dynamics) Mode Imager (Amersham Biosciences). Steady-state Kinetics of ABH2 and ABH3—Determination of the kcat and Km values of the ABH2 and ABH3 was made by varying the substrate concentration (10, 20, 50, 100, 200, and 400 nm). The substrates used were the same ODNs as those in the previous section. Both ss- and ds-DNA substrates were used. The total protein concentration in each case was 5 pm. Reactions were performed for 60 min or adjusted to ensure that less than 20% of the substrate was expended. All reactions were performed in triplicate and analyzed using Hanes plots according to Equation 1, [ODNTotal]/v=[ODNTotal]/Vmax+Km/Vmax(Eq. 1) where ODNtotal is the total ODN substrate concentration, Vmax is the enzyme maximum reaction velocity, v is the initial reaction velocity, and Km is the Michaelis constant. Reactions of ds-DNA substrates with ABH2 were performed in 150 mm NaCl in the reaction buffer based on results with respect to dependence on salt concentration. NMR Studies—The wild-type and mutant ABH2 and ABH3 proteins were characterized by one-dimensional NMR experiments to ensure that the amino acid substitutions do not disrupt the overall structural integrity of the protein. The samples for NMR measurements were at ∼1 mg/ml concentration, in 10 mm sodium phosphate buffer in 95% H2O, 5% D2Oat pH 6.0. One-dimensional 1H experiments were performed on a Bruker Avance 500 MHz spectrometer equipped with four channels, pulse shaping, and pulsed field gradient capabilities. The spectra were collected with 8192 complex points and 8000 Hz spectral width. Northern Blot Analysis—Human or mouse multiple-tissue poly(A)+ Northern blots were obtained from Clontech. The membranes were hybridized with a probe containing the complete cDNA of human or mouse AlkB homologues. A probe specific for β-actin was used as a control. Membranes were exposed overnight to storage phosphor screens and were scanned at 200 micron resolution using a Typhoon 9410 Variable Mode Imager (Amersham Biosciences). Signal quantification was performed using ImageQuant 5.2 software (Molecular Dynamics). Cloning and Sequence Alignment of mABH2 and mABH3—Mouse homologues of human ABH2 and 3, mAbh2 and mAbh3, were cloned by homology to the corresponding human proteins. The alignment of the proteins using ClustalW is shown in Fig. 1c. The identity between the mouse and human proteins suggests that the function should be conserved in these derivatives. The identity between mAbh2 and ABH2 is over 75.1%, but that is due mainly to differences in the N-terminal end of the proteins. In contrast, the identity for mAbh3 and ABH3 is 85.7% with the majority of bases that differ spread throughout the two sequences. There are a number of sequences that are conserved for all five sequences in Fig. 1c. These sequences indicated in yellow could have structural or catalytic roles. Among these, there is a cluster that has H separated by an amino acid and DDE (for ABH2, D173, Fig. 1c). This region could have a role in catalysis. The AlkB protein is very close in its sequence alignment in this region with the only difference being a lysine substituted in the sequence to separate the two consecutive aspartic acids. These alignments are close, but functional assays on the proteins must be performed to indicate that the proteins have the same activities. Interestingly, there is only 27% identity between mAbh2 and mAbh3. The identity drops to 19.1 or 12.6% when the AlkB is compared independently to mAbh2 or mAbh3. Even though the identity in bioinformatics alignments involving mAbh2 and mAbh3 with AlkB is low, both proteins still are active against similar substrates (see below). Expression and Purification of mAbh2 and mAbh3—To study the biochemical properties of mAbh2 and mAbh3, we expressed the mAbh2 and mAbh3 proteins using a T7 RNA polymerase promoter-based system in E. coli HK82(DE3) (alkB-) with His6 tags to use Ni-NTA chromatography in the initial purification step. Further purification of the proteins was performed using CM-Sepharose and gel filtration chromatography to obtain homogeneous proteins (Fig. 2a). To compare the proteins to their human counterparts, the ABH2 and ABH3 were isolated using similar methods. To ensure that the major peaks from the final columns corresponded to the activity of the peaks observed for the proteins, we monitored the activity against a number of 3H-labeled substrates (Fig. 2b). Both mAbh2 and mAbh3 (not shown) purification demonstrated that the major peaks obtained in the final purification were the major activities obtained. Expression of the ABH2, ABH3, mAbh2, and mAbh3 produced more than 10 mg/liter of purified protein. mAbh2 and mAbh3 Reactions Complement Sensitivity of E. coli alkB-Strain HK82 to MMS-treated M13mp18—To test the activities of the purified proteins and their capacity to complement an alkB-deficient E. coli strain, ss-M13mp18 DNA was treated with MMS, the methylated ss-DNA treated with one of the AlkB homologue proteins, and transformed into an alkB strain (HK82) or as a control, a wild-type strain (HK81) (Fig. 3). If the methylated ss-DNA is not reacted with one of the AlkB homologue proteins, the transformation efficiency in the HK82 alkB- strain as determined by plaque formation is almost two orders of magnitude less than when the modified ss-DNA is treated with an AlkB homologue prior to transformation. In this assay, transformation efficiency is determined by the number of plaques formed compared with unmodified ss-M13mp18 DNA. The modified ss-DNA was transformed into the wild-type strain, HK81, without reaction with any of the AlkB homologues and demonstrated to have similar transformation efficiency to that of the modified ss-DNA treated with AlkB homolgues, but transformed into the HK82 alkB-deficient strain. Any of the AlkB homologues (ABH2, ABH3, mAbh2, or mAbh3) used for treatment of modified ss-DNA yielded transformation efficiencies comparable to that for transformation into the wild-type HK81 strain. Therefore, the mAbh2 and mAbh3 have similar activities to ABH2 and ABH3 that restore the transformation efficiency of methylated ss-DNA. mAbh2 and mAbh3 Remove Methyl Groups from 1-meA and 3-meC—The AlkB homologues repair the alkylated base via an oxidative mechanism that releases formaldehyde and does not involve scission of the phosphodiester backbone (17Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (634) Google Scholar, 22Aas P.A. Otterlei M. Falnes P.O. Vagbo C.B. Skorpen F. Akbari M. Sundheim O. Bjoras M. Slupphaug G. Seeberg E. Krokan H.E. Nature. 2003; 421: 859-863Crossref PubMed Scopus (521) Google Scholar, 25Margison G. DNA Repair (Amst.). 2002; 1: 1057-1061Crossref PubMed Scopus (7) Google Scholar, 26Sedgwick B. Lindahl T. Oncogene. 2002; 21: 8886-8894Crossref PubMed Scopus (116) Google Scholar, 27Begley T.J. Samson L.D. Trends Biochem. Sci. 2003; 28: 2-5Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). To study the release of methyl groups by the 2OG activity of AlkB homologues, we used an assay based on ODNs with unique modifications of 1-meA or 3-meC in restriction enzyme cleavage sites. The modified ODN is first treated with the AlkB homologue. If ss, the ODN must be annealed to the complementary ODN prior to restriction enzyme cleavage. The ds-ODN is then reacted with a restriction enzyme and the ODN is cleaved if the methyl group on 1-meA or 3-meC has been removed. Following restriction endonuclease cleavage, the products are separated using denaturing PAGE. Fig. 4 shows results from the use of this assay for ss-DNA substrates. If there is no modified ODN, all DNA is cleaved by EcoRI. However if a 1-meA is present in the first A in the EcoRI restriction site, the DNA is not cleaved by the restriction enzyme. Partial removal of the 1-meA by the ABH2, ABH3, mAbh2, or mAbh3 restores the restriction site and permits cleavage by EcoRI. To test the universality of this assay, we also built a 1-meA ODN with a HindIII site and placed the 1-meA at the first A of the recognition sequence. In this case, no inhibition of the cleavage is observed (data not shown). Therefore, this is a useful assay, but not every restriction site can be employed. An assay to monitor 3-meC activity based on the same principles was also developed. In this case, 3-meC was inserted into an ODN at the first C position of a HpaII site. The same procedure was followed to demonstrate activity for the 1-meA assay, except that HpaII restriction endonuclease cleavage was used. Fig. 5 unambiguously demonstrates that there is only cleavage of the ODN when 3-meC has been removed from the HpaII site. We also tested KpnI and HhaI restriction sites in a similar assay, but found that KpnI and HhaI cleavage, although inhibited, did not yield clear results as did use of the HpaII site (data not shown). The use of this assay method should greatly simplify the determination of kinetics and the study of AlkB homologues. The structural integrity of mutant AlkB proteins was evaluated by one-dimensional NMR spectra. The one-dimensional NMR spectra of the wild-type and mutant proteins were nearly identical, particularly in the region of the spectra corresponding to signals of methyl and methylene groups, which include those from amino acid residues in the hydrophobic core of the protein, such as Ile, Leu, and Val. The one-dimensional spectra indicate that the hydrophobic core of the protein is intact in the mutants, and therefore, the amino acid substitutions do not disrupt the overall three-dimensional structure of the protein (data not shown). ABH2 and mAbh2 Remove Methyl Groups from Single-stranded RNA and DNA Substrates—The AlkB homologues were tested against a number of substrates for activities removing methylation damage. The substrates were ODNs, polydeoxyribonucleotides, or polyribonucleotides that were modified using [3H]MNU. The rate of release of radioactivity is similar for mAbh2 and ABH2 for ODNs, either single- or double-stranded that are modified with [3H]MNU (Fig. 6, a and c). By contrast, mAbh3 and ABH3 release radioactivity from ss-ODNs much faster than from ds-ODNs (Fig. 6, b and d). The ss-polydeoxyribonucleotides, ss-polyribonucleotides, and M13mp18 were also examined for the release of radioactivity. For mAbh2 and ABH2, there are again similarities. Release of the radioactivity from [3H]CH3-poly(dA) or [3H]CH3-poly(dC) is at least 10-50-fold more rapid than release of radioactivity from [3H]CH3-poly(A) or [3H]CH3-poly(C) (Fig. 6, e and g). We also examined the release of radioactivity from [3H]CH3-M13mp18 and found that less than 10% of the total radioactivity in the assay was released by mAbh2 or ABH2 treatment. However, release of radioactivity from the [3H]CH3-M13mp18 substrate by mAbh2 was significantly lower than for ABH2. Thus, the mAbh2 removes 1-meA or 3-meC from both RNA and DNA, but there is a distinct bias for the removal from DNA. For mAbh3 and ABH3, release of methyl groups from polydeoxyribonucleotide and polyribonucleotide substrates had similar efficiencies, showing that mAbh3 and ABH3 recognize RNA and DNA (Fig. 6, f and h). It is also possible that the mAbh2, mAbh3, ABH2, or ABH3 could harbor activities associated with exo or endonucleases that would release radioactivity in the assays. To show that the activities are not associated with exo or endonucleases, we end-labeled either an ODN or a polyribonucleotide and treated these substrates with the enzymes. Only minor amounts of radioactivity were released by the purified AlkB homologues (see supplementary data) from the 3′-end of the ODN. A control using exonuclease I, a 3′ → 5′ exonuclease, shows that the pattern expected for the degradation for that type of exonuclease. No degradation of the ODN by the AlkB homologues is observed, consistent with the absence of exo or endonuclease activity. The fact that the intensities of the full-length ODNs that are 5′-end-labeled do not change, argues that there is no 5′ → 3′ exonuclease. In another experiment, poly(C) RNA was labeled using T4 polynucleotide kinase. Again, no degradation of the ribonucleotide is observed, suggesting that the activity releasing tritium into the medium is not caused by exonuclease degradation of polyribonucleotides (supplementary data). We also modified ODNs with unlabeled DMS and monitored the release of formaldehyde using the Nash assay. This demonstrated that the reaction of the four AlkB homologues all released formaldehyde (data not shown). The ensemble of the experiments on the DNA substrates shows that the mAbh2 and mAbh3 release methyl groups from DNA using a mechanism similar to that of AlkB or the ABH2 or ABH3. ABH2 and ABH3 Have Different Salt
Referência(s)