Minimal Methylated Substrate and Extended Substrate Range of Escherichia coli AlkB Protein, a 1-Methyladenine-DNA Dioxygenase

The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of α-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5′-phosphorylated 1-me-dAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5′ phosphate group was required. Consequently, 1-me-dAMP(5′) was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-me-dATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-me-dATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts. The Escherichia coli AlkB protein, and two human homologs ABH2 and ABH3, directly demethylate 1-methyladenine and 3-methylcytosine in DNA. They couple Fe(II)-dependent oxidative demethylation of these damaged bases to decarboxylation of α-ketoglutarate. Here, we have determined the kinetic parameters for AlkB oxidation of 1-methyladenine in poly(dA), short oligodeoxyribonucleotides, nucleotides, and nucleoside triphosphates. Methylated poly(dA) was the preferred AlkB substrate of those tested. The oligonucleotide trimer d(Tp1meApT) and even 5′-phosphorylated 1-me-dAMP were relatively efficiently demethylated, and competed with methylated poly(dA) for AlkB activity. A polynucleotide structure was clearly not essential for AlkB to repair 1-methyladenine effectively, but a nucleotide 5′ phosphate group was required. Consequently, 1-me-dAMP(5′) was identified as the minimal effective AlkB substrate. The nucleoside triphosphate, 1-me-dATP, was inefficiently but actively demethylated by AlkB; a reaction with 1-me-ATP was even slower. E. coli DNA polymerase I Klenow fragment could employ 1-me-dATP as a precursor for DNA synthesis in vitro, suggesting that demethylation of alkylated deoxynucleoside triphosphates by AlkB could have biological significance. Although the human enzymes, ABH2 and ABH3, demethylated 1-methyladenine residues in poly(dA), they were inefficient with shorter substrates. Thus, ABH3 had very low activity on the trimer, d(Tp1meApT), whereas no activity was detected with ABH2. AlkB is known to repair methyl and ethyl adducts in DNA; to extend this substrate range, AlkB was shown to reduce the toxic effects of DNA damaging agents that generate hydroxyethyl, propyl, and hydroxypropyl adducts. Alkylating agents occur in the environment and arise endogenously during cellular metabolism. They damage DNA at multiple sites, and the lesions generated may result in mutagenesis and cell death. The importance of preventing these adverse effects is highlighted by the variety of DNA repair mechanisms that have evolved to remove alkylated bases from DNA. These repair functions are conserved from bacteria to humans. In Escherichia coli, the activities are induced up to 1000-fold (1Sedgwick B. Lindahl T. Oncogene. 2002; 21: 8886-8894Crossref PubMed Scopus (116) Google Scholar) in the Ada response (the adaptive response to alkylating agents). 3-Methyladenine-DNA glycosylases excise cytotoxic 3-methyladenine and related lesions from DNA in the first step of base excision repair (2Hollis T. Lau A. Ellenberger T. Mutat. Res. 2000; 460: 201-210Crossref PubMed Scopus (64) Google Scholar). In contrast, suicidal O 6-methylguanine-DNA methyltransferases directly demethylate the highly mutagenic and toxic lesion O 6-methylguanine by transferring the methyl group onto a cysteine residue in the protein (3Daniels D.S. Tainer J.A. Mutat. Res. 2000; 460: 151-163Crossref PubMed Scopus (83) Google Scholar). A third type of DNA repair mechanism specific for alkylated bases was recently resolved. The E. coli AlkB protein (4Kataoka H. Yamamoto Y. Sekiguchi M. J. Bacteriol. 1983; 153: 1301-1307Crossref PubMed Google Scholar) and its human homologs, ABH2 and ABH3, oxidize the methyl groups of 1-methyladenine (1-meA) 1The abbreviations used are: 1-meA, 1-methyladenine; 3-meC, 3-methylcytosine; DMS, dimethyl sulphate; MeI, methyl iodide; EtI, ethyl iodide; PrI, propyl iodide; HPLC, high pressure liquid chromatography. and 3-methylcytosine (3-meC) in DNA to directly regenerate unmodified adenine and cytosine residues. The methyl adduct is released as formaldehyde (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar, 6Falnes P.O. Johansen R.F. Seeberg E. Nature. 2002; 419: 178-181Crossref PubMed Scopus (508) Google Scholar, 7Duncan 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 (316) Google Scholar, 8Aas 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 (526) Google Scholar). AlkB and its human counterparts are members of the α-ketoglutarate/Fe(II)-dependent dioxygenase superfamily (9Aravind, L., and Koonin, E. V. (2001) Genome Biology, 2,0007.1–0007.8Google Scholar), and couple decarboxylation of α-ketoglutarate to oxidative demethylation of the damaged bases. The lesions 1-meA and 3-meC are formed mainly in single-stranded DNA (10Bodell W.J. Singer B. Biochemistry. 1979; 18: 2860-2863Crossref PubMed Scopus (38) Google Scholar) and are predicted to arise at replication forks and in actively transcribed genes where they could block DNA and RNA polymerases (11Boiteux S. Laval J. Biochimie (Paris). 1982; 64: 637-641Crossref PubMed Scopus (41) Google Scholar, 12Larson K. Sahm J. Shenkar R. Strauss B. Mutat. Res. 1985; 150: 77-84Crossref PubMed Scopus (193) Google Scholar). Indeed, AlkB, ABH2, and ABH3 repair these lesions in single-stranded DNA, but also in oligonucleotides that have been annealed with a complementary strand after alkylation (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar, 6Falnes P.O. Johansen R.F. Seeberg E. Nature. 2002; 419: 178-181Crossref PubMed Scopus (508) Google Scholar, 7Duncan 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 (316) Google Scholar, 8Aas 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 (526) Google Scholar,13Dinglay S. Trewick S.C. Lindahl T. Sedgwick B. Genes Dev. 2000; 14: 2097-2105PubMed Google Scholar). ABH2 is exclusively a nuclear enzyme, whereas overexpressed ABH3 is detected both in the nucleus and cytoplasm (7Duncan 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 (316) Google Scholar, 8Aas 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 (526) Google Scholar). Methylating agents are probably the major source of endogenous DNA alkylation; however, other types of alkylating agents occur in the environment and may generate larger adducts. We have shown previously that AlkB protects against the toxicity of ethylation damage and reverts 1-ethyladenine to adenine in DNA yielding acetaldehyde as a reaction product (7Duncan 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 (316) Google Scholar). Ethylene oxide, a known mutagen and carcinogen, is formed endogenously during ethylene metabolism and is also widely used as a fumigant for sterilization. Hydroxyethyl adducts generated by this compound have been detected in cellular DNA (14Kumar R. Hemminki K. Carcinogenesis. 1996; 17: 485-492Crossref PubMed Scopus (35) Google Scholar). Other small alkylating epoxides are also employed in large quantities in the chemical industry (15Melnick R.L. Ann. N. Y. Acad. Sci. 2002; 982: 177-189Crossref PubMed Scopus (72) Google Scholar). Oligonucleotides containing defined single adducts have been used in kinetic, mechanistic, and structural studies of DNA repair enzymes. In this paper, we have examined the ability of E. coli AlkB, and human ABH2 and ABH3 to repair 1-meA in oligodeoxynucleotides, nucleotides, and nucleoside triphosphates. Substrate characteristics that influence the efficiency of repair have been defined, and the minimal substrate that is demethylated effectively by AlkB has been determined. To extend the known substrate range of AlkB to other alkyl adducts induced by environmental agents, we have also examined the ability of AlkB to counteract the adverse effects of small epoxides and larger alkyl halides. Materials—[14C]MeI (58 mCi/mmol), [γ-32P]ATP (3000 Ci/mmol), and poly(dA) (average chain length 310 residues) were obtained from Amersham Biosciences. Oligonucleotides were prepared on an Applied Biosystems 3948 synthesizer by the Cancer Research UK oligonucleotide synthesis facility. Other reagents were from Sigma-Aldrich. Preparation of Substrates—[14C]MeI-treated poly(dA) was prepared as described previously (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar, 7Duncan 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 (316) Google Scholar). Non-radioactive substrates were made by repeatedly exposing oligonucleotides, mononucleotides, and deoxyadenosine in 100 mm ammonium acetate, pH 6.5, to a 10-fold molar excess of dimethylsulphate (DMS) every 2–3 h for 24 h at 20 °C. The pH of the reaction was maintained between 4 and 7 by adding NaOH when necessary (16Singer B. Sun L. Fraenkel-Conrat H. Biochemistry. 1974; 13: 1913-1920Crossref PubMed Scopus (57) Google Scholar). Products methylated at N1-adenine were purified by reverse phase HPLC using a 2 × 150 mm Phenomenex Luna C-18 (2Hollis T. Lau A. Ellenberger T. Mutat. Res. 2000; 460: 201-210Crossref PubMed Scopus (64) Google Scholar) column and Beckman System Gold. The flow rate was 0.2 and a of in mm ammonium acetate, pH 6.5, was Products were at A In the methylated product the and was clearly resolved. purified 1-me-dATP, of was prepared by the 1-me-dATP and this To the purified methylated a was in at for 20 and the released 1-meA by reverse phase HPLC and A A of was between and by between and coli AlkB protein with an was purified as previously described (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar, S. Trewick S.C. Lindahl T. Sedgwick B. Genes Dev. 2000; 14: 2097-2105PubMed Google Scholar). The is by a from the of the AlkB protein The was using AlkB was with at 20 for was to the at 4 to the released and for protein was by of the was by The for AlkB, and also for ABH2 and ABH3, were as previously described for and were at and were not in the (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar, 7Duncan 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 (316) Google Scholar). To and the were at of the methylated substrates. The were with substrate molar in a for to at °C. on poly(dA) was by the of (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar). using were by adding to The substrates were by at for 20 The adenine released was by The reaction was determined from the adenine generated and were from K. K. J. and of Biochemistry. Scholar) of the were at with and are for In DNA oligonucleotide and the were purified by The was with [γ-32P]ATP using polynucleotide and purified a The and were in in mm pH mm and annealed by to for by to 20 °C. were using DNA polymerases and in the of polymerase was to a reaction containing of deoxynucleoside and annealed substrate. for at °C. The reaction were in of and by were detected by of the of and for of ABH2 and ABH3 in the ABH2 ABH3 were using of ABH2 and ABH3 the has been described (7Duncan 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 (316) Google Scholar). were from to using These were E. coli and bacteria DNA containing ABH2 ABH3 using and were to DNA was and to the containing were to generate an of ABH2 and ABH3 from were with at a of of and for when ABH2 and for 4 when were in of containing mm pH 150 mm and mm 20 on cellular was by at × at 4 °C. were onto a of the with column of but containing mm and using but containing mm were using an and was to for ABH2 and for of single-stranded DNA were with alkylating and propyl were in and to 100 of to the The and of with the agents are in of alkylated E. coli and was as previously described S. Trewick S.C. Lindahl T. Sedgwick B. Genes Dev. 2000; 14: 2097-2105PubMed Google Scholar). of 1-meA in Oligonucleotides and by was to AlkB could repair 1-meA in short oligonucleotides and To the and and adenine were methylated by to DMS, that of the adenine residues in the were to The methylated were purified by reverse phase AlkB protein was with purified substrates in (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar). for of methylated trimer was directly to unmodified by AlkB and also but inefficiently by the human ABH3 protein and of AlkB on and AlkB demethylated the and also 1-me-dAMP and 1-me-dATP of repair of alkylated substrates by methylated in a To the efficiency of repair of 1-meA in AlkB was using of the and the and from of the of poly(dA) was by of other substrates containing 1-meA, repair was by the of adenine of are shown in AlkB activity on and The kinetic determined for repair of substrates are in The a of the of the substrates. the substrates methylated poly(dA) was the with methylated poly(dA), the of repair of the trimer d(Tp1meApT), and the with both and of AlkB for these substrates. The was a very substrate may the of a phosphate residue 5′ to 1-meA in this In with this AlkB demethylated 5′-phosphorylated 1-me-dAMP efficiently 5′ of using polynucleotide its to demethylation by AlkB not Thus, a phosphate residue 5′ to the lesion the of AlkB for AlkB had but activity on using a 10-fold excess of substrate enzyme, of the methylated deoxynucleoside was to the in not 1-me-dATP was actively demethylated by AlkB, but the efficiency was for 1-me-dAMP(5′) of of the methylated deoxynucleoside is of the of nucleoside triphosphates are larger those of mononucleotides, they a larger for The triphosphate, could also demethylated by AlkB, but even efficiently 1-me-dATP To the AlkB the was by The protein had the as the protein for activity on both methylated poly(dA) and d(Tp1meApT), the were to not The activity of AlkB on the substrates was by the between and protein to single-stranded DNA, and efficiently to the methylated S. Trewick S.C. Lindahl T. Sedgwick B. Genes Dev. 2000; 14: 2097-2105PubMed Google Scholar). The single-stranded substrates in a every base residue for damage by the substrate Y. J. 2003; PubMed Scopus Google Scholar). The of with substrates To that repair of is the using and substrates were of the 1-me-dAMP(5′) were to the AlkB reaction containing poly(dA), in 4 in residues were of the in a rate of of a to excess of the substrates was to a in the rate of reaction with the the a the These the of AlkB for a substrate. of 1-me-dAMP by the Klenow of E. coli DNA of 1-me-dATP by AlkB a for AlkB in this damaged nucleoside from the DNA precursor of biological 1-me-dATP used as a substrate by DNA A that 1-me-dATP was during in DNA synthesis by DNA however, a of 1-me-dATP and was used in those Nature. 1982; PubMed Scopus Google Scholar). To that 1-me-dATP used as a DNA 1-me-dATP was purified and of 1-me-dAMP by E. coli polymerase I Klenow fragment during in DNA synthesis The 1-me-dATP was purified from and by HPLC and by and HPLC The 1-me-dATP was to A 5′ oligonucleotide was annealed to a and by the Klenow fragment in the of of deoxynucleoside triphosphates In the of 1-me-dATP Klenow fragment 5′ a single residue to the a base in the The 1-me-dAMP could by the polymerase when and were Thus, 1-me-dAMP was both and by Klenow a of the Klenow of 1-me-dAMP were at residue not an of was also the of the polymerase were no Thus, 1-me-dATP to employed for DNA synthesis In to the Klenow with low 5′ activity human DNA polymerase a 1-me-dAMP residue at the not of AlkB on and human homologs, ABH2 and ABH3, of the E. coli AlkB protein were previously identified (7Duncan 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 (316) Google Scholar, 8Aas 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 (526) Google Scholar). have the as AlkB, and repair 1-meA and 3-meC in DNA. The of ABH2 and ABH3 were previously with to pH and activities of the in were and of AlkB when repair of 1-meA in The two with had been purified after in E. coli (7Duncan 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 (316) Google Scholar). To in the ABH2 and ABH3 were ABH2 and ABH3 purified from and when purified from E. coli, activities and of AlkB activity on methylated purified protein were by but no were detected not The activity of ABH2 and ABH3 purified from may result from of the overexpressed To the human were on and ABH2 and ABH3 purified from were with the trimer d(Tp1meApT) and ABH3 was to demethylate d(Tp1meApT), but the activity was very low of ABH2 on d(Tp1meApT) was not of ABH2 ABH3 on 1-me-dATP not of AlkB on in are in of with and that AlkB both methyl and ethyl adducts generated in single-stranded DNA (7Duncan 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 (316) Google Scholar, S. Trewick S.C. Lindahl T. Sedgwick B. Genes Dev. 2000; 14: 2097-2105PubMed Google Scholar). We have used the as an of AlkB repair other types of alkylation damage larger alkyl and bases. In this the known substrate range of AlkB may was with EtI, PrI, agents, and in an and its The of was examined previously (7Duncan 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 (316) Google Scholar), but is for with the other alkylating The of both and was in the that AlkB both and DNA bases DNA and are induced by the alkylating as ethylene and F. A. PubMed Scopus Google Scholar). to the and of ethylene oxide, we used as a compound to DNA lesions S. Nature. PubMed Scopus Google Scholar), and to generate hydroxypropyl adducts with oxide, was in the in the that AlkB both and hydroxypropyl base adducts (7Duncan 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 (316) Google Scholar). Substrate for AlkB activity have been by the ability of this to demethylate 1-meA residues in oligonucleotides, and The substrate of those examined was methylated 1-meA residues the trimer d(Tp1meApT), and in the were efficiently We that these low substrates the structural essential for AlkB to to its substrate and 1-meA in its Thus, a polynucleotide with is not for demethylation but may result in of 1-meA for contrast, substrates were those a phosphate 5′ to the the and E. coli the of a 5′ phosphate to excise a 5′ residue from a minimal substrate U. Biochemistry. PubMed Scopus Google Scholar). In a J. 2003; PubMed Scopus Google Scholar), the methylated and were not demethylated by AlkB, they were by the AlkB and AlkB of an reaction of α-ketoglutarate with the methyl group on these is in the and to efficiently with the Here, we very demethylation of by with a 5′ phosphate on the substrate may to the methyl group for We have identified 1-me-dAMP(5′) as the minimal effective AlkB substrate. The of d(Tp1meApT) and 1-me-dAMP(5′) as low but effective substrates of AlkB containing a single defined adduct may of in of the structure of the AlkB of the of AlkB to its substrate necessary to and extend the on substrate In this we have by that and at the of the protein, but are T. and B. DNA polymerase I Klenow fragment was shown to and extend 1-me-dAMP residues using a 1-me-dATP precursor during DNA synthesis in vitro, that was The to and could occur in on the DNA polymerases they 1-me-dAMP residues and extend from the by 1-meA at the In this may of that human DNA polymerase could not 1-me-dATP in in A for this is the ability of the Klenow but not polymerase to that are to with the Proc. Natl. Acad. Sci. U. S. A. 2002; Scopus Google Scholar). of 1-me-dATP by AlkB could its for DNA synthesis in by I in E. Other that on damaged DNA are the E. coli and and human and to of these mutagenic and toxic DNA H. Sekiguchi M. Nature. PubMed Scopus Google Scholar, B. J. Bacteriol. PubMed Google Scholar). AlkB of E. coli not a on with a S. Trewick S.C. Lindahl T. Sedgwick B. Genes Dev. 2000; 14: 2097-2105PubMed Google Scholar), of 1-me-dAMP residues to a to of and residues during DNA The AlkB could demethylate both 1-me-dATP and but the was a substrate has been shown previously that DNA repair by damage also repair Thus, RNA is by a in of to regenerate biological activity of the RNA J. 1974; PubMed Scopus Google Scholar). The DNA from E. coli not in DNA, but also in and an RNA as A. Biochemistry. PubMed Scopus Google Scholar). the O 6-methylguanine-DNA DNA alkylation directly demethylate O 6-methylguanine residues in RNA P. Proc. Natl. Acad. Sci. U. S. A. 1985; PubMed Scopus Google Scholar, K. PubMed Scopus Google Scholar). These on RNA repair have to the are and those with damaged DNA. 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. 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A. 2002; 99: 16660-16665Crossref PubMed Scopus (316) Google Scholar, 8Aas 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 (526) Google Scholar). an that the of single-stranded RNA in cell could a for toxic alkylating agents in of and in the of however, is the RNA repair by AlkB and ABH3 are of with to of and the repair of 1-me-ATP by AlkB is of biological significance. the of alkylated in an E. coli we have the AlkB substrate range to propyl, hydroxyethyl, and hydroxypropyl DNA adducts. The methylated and DNA bases that are are and (5Trewick S.C. Henshaw T.F. Hausinger R.P. Lindahl T. Sedgwick B. Nature. 2002; 419: 174-178Crossref PubMed Scopus (638) Google Scholar, 7Duncan 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 (316) Google Scholar), and this is also the for the propyl and adducts. is efficiently The in in the and its was after with the agents after with of lesions by AlkB may even that of ethyl and propyl other toxic lesions may formed that are not substrates of DNA glycosylases and DNA methyltransferases that repair methyl adducts also repair larger alkyl adducts with a efficiency Res. PubMed Scopus Google Scholar, B. Laval J. Mutat. Res. PubMed Scopus Google Scholar). with glycosylases and O we that AlkB and the human ABH2 and ABH3 are to as these DNA repair on DNA but to the major substrate identified in in DNA is substrate of AlkB and its homologs, the repair could also a a when required. We for and for with protein

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