Repair of Formamidopyrimidines in DNA Involves Different Glycosylases

Abstract

The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylase-deficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions. The oxidatively induced DNA lesions 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA) are formed abundantly in DNA of cultured cells or tissues exposed to ionizing radiation or to other free radical-generating systems. In vitro studies indicate that these lesions are miscoding, can block the progression of DNA polymerases, and are substrates for base excision repair. However, no study has yet addressed how these lesions are metabolized in cellular extracts. The synthesis of oligonucleotides containing FapyG and FapyA at defined positions was recently reported. These constructs allowed us to investigate the repair of Fapy lesions in nuclear and mitochondrial extracts from wild type and knock-out mice lacking the two major DNA glycosylases for repair of oxidative DNA damage, OGG1 and NTH1. The background level of FapyG/FapyA in DNA from these mice was also determined. Endogenous FapyG levels in liver DNA from wild type mice were significantly higher than 8-hydroxyguanine levels. FapyG and FapyA were efficiently repaired in nuclear and mitochondrial extracts from wild type animals but not in the glycosylase-deficient mice. Our results indicated that OGG1 and NTH1 are the major DNA glycosylases for the removal of FapyG and FapyA, respectively. Tissue-specific analysis suggested that other DNA glycosylases may contribute to FapyA repair when NTH1 is poorly expressed. We identified NEIL1 in liver mitochondria, which could account for the residual incision activity in the absence of OGG1 and NTH1. FapyG and FapyA levels were significantly elevated in DNA from the knock-out mice, underscoring the biological role of OGG1 and NTH1 in the repair of these lesions. A large number of DNA base modifications are formed by oxidative damage to DNA (for review, see Ref. 1Evans M.D. Dizdaroglu M. Cooke M.S. Mutat. Res. 2004; 567: 1-61Crossref PubMed Scopus (1057) Google Scholar). Some of these lesions are generated at high rates, even in the absence of exogenous DNA-damaging agents. For instance, it was estimated that 100–500 8-hydroxyguanines (8-oxoGs) 3The abbreviations used are: 8-oxoG8-hydroxyguanine8-oxo-dG8-hydroxy-2′-deoxyguanosineOGG1oxoguanine DNA glycosylaseNTH1endonuclease III homologueFapyG2,6-diamino-4-hydroxy-5-formamidopyrimidineFapyA4,6-diamino-5-formamidopyrimidineMe-FapyG2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidineMLMEmouse liver mitochondrial extractsMLNEmouse liver nuclear extractsTgthymine glycolWTwild typeLC/MSliquid chromatography/mass spectrometryGC/MSgas chromatography/mass spectrometry. are formed per day in a human cell (2Lindahl T. Nature. 1993; 362: 709-715Crossref PubMed Scopus (4378) Google Scholar). 8-oxoG is one of the most studied DNA lesions, and it is often used as a biomarker of oxidative DNA damage. However, ring-opened formamidopyrimidine lesions, 2,6-diamino-4-hydroxy-5-formamidopyrimidine (FapyG) and 4,6-diamino-5-formamidopyrimidine (FapyA), are formed at equal or higher levels than 8-oxoG after oxidative stress (3Dizdaroglu M. Olinski R. Doroshow J.H. Akman S.A. Cancer Res. 1993; 53: 1269-1272PubMed Google Scholar, 4Mori T. Hori Y. Dizdaroglu M. Int. J. Radiat. Biol. 1993; 64: 645-650Crossref PubMed Scopus (62) Google Scholar, 5Kasprzak K.S. Jaruga P. Zastawny T.H. North S.L. Riggs C.W. Olinski R. Dizdaroglu M. Carcinogenesis. 1997; 18: 271-277Crossref PubMed Scopus (57) Google Scholar). These lesions result from hydroxyl radical attack on guanine and adenine, respectively, followed by one-electron reduction of the hydroxyl adduct radicals (1Evans M.D. Dizdaroglu M. Cooke M.S. Mutat. Res. 2004; 567: 1-61Crossref PubMed Scopus (1057) Google Scholar), which are also intermediates in the formation of 8-oxoG and 8-oxoA (6Candeias L.P. Steenken S. Chemistry. 2000; 6: 475-484Crossref PubMed Scopus (31) Google Scholar). Formation of Fapy lesions in DNA upon UV radiation has also been reported (7Doetsch P. Zastawny T. Martin A. Dizdaroglu M. Biochemistry. 1997; 34: 737-742Crossref Scopus (110) Google Scholar). 8-hydroxyguanine 8-hydroxy-2′-deoxyguanosine oxoguanine DNA glycosylase endonuclease III homologue 2,6-diamino-4-hydroxy-5-formamidopyrimidine 4,6-diamino-5-formamidopyrimidine 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine mouse liver mitochondrial extracts mouse liver nuclear extracts thymine glycol wild type liquid chromatography/mass spectrometry gas chromatography/mass spectrometry. FapyA (8Delaney M.O. Wiederholt C.J. Greenberg M.M. Angew. Chem. Int. Ed. Engl. 2002; 41: 771-773Crossref PubMed Scopus (53) Google Scholar) and FapyG (9Wiederholt C.J. Greenberg M.M. J. Am. Chem. Soc. 2002; 124: 7278-7279Crossref PubMed Scopus (86) Google Scholar) are miscoding in vitro, both directing the preferential misincorporation of adenine opposite the lesions by a bacterial DNA polymerase (Klenow exo-). Experiments using the methylated analogue of FapyG, i.e. 2,6-diamino-4-hydroxy-5-N-methylformamidopyrimidine (Me-FapyG), have suggested that formamidopyrimidines might also constitute blocks to DNA polymerases (10Graziewicz M.A. Zastawny T.H. Olinski R. Speina E. Siedlecki J. Tudek B. Free Radic. Biol. Med. 2000; 28: 75-83Crossref PubMed Scopus (32) Google Scholar, 11Asagoshi K. Terato H. Ohyama Y. Ide H. J. Biol. Chem. 2002; 277: 14589-14597Abstract Full Text Full Text PDF PubMed Scopus (44) Google Scholar). It is noteworthy, however, that FapyG and FapyA are chemically distinct from Me-FapyG. FapyG and FapyA are formed from purines in DNA by oxidative attack or UV radiation, whereas Me-FapyG is the product of alkylation damage to guanine in DNA. Consequently, any results obtained with Me-FapyG should not be directly extrapolated to FapyG or FapyA. Oxidative DNA damage is primarily repaired by the base excision repair pathway. Base excision repair is initiated by a DNA glycosylase that recognizes the modified base and hydrolyzes the N-glycosidic bond. Repair then proceeds through the coordinated actions of an abasic site endonuclease, DNA polymerase, and DNA ligase (12Wilson D.M. II I. Sofinowski T.M. McNeill D.R. Front Biosci. 2003; 8: d963-d981Crossref PubMed Google Scholar). Numerous DNA glycosylases have been identified in mammals, and although they show distinct substrate specificities, there is a considerable degree of overlap, most notably among DNA glycosylases that repair oxidative DNA damage (for review, see Ref. 13Dizdaroglu M. Mutat. Res. 2003; 531: 109-126Crossref PubMed Scopus (75) Google Scholar). Studies with purified enzymes from various sources have identified several glycosylases that can release FapyG and FapyA from irradiated DNA, such as the bacterial Fpg, Nei, and Nth; the yeast, plant, and mammalian oxoguanine DNA glycosylase (OGG1); and the recently identified human and mouse homologues of Escherichia coli endonuclease VIII (Nei) (NEIL1) (14Hazra 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, 15Jaruga P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (80) Google Scholar). However, to date, no study has directly addressed repair of FapyG and FapyA in cellular extracts from mammalian cells. Synthetic single lesion oligonucleotide constructs have been instrumental for the understanding of repair pathways for modified DNA bases (16Wang D. Kreutzer D.A. Essigmann J.M. Mutat. Res. 1998; 400: 99-115Crossref PubMed Scopus (428) Google Scholar). A method for the synthesis of oligonucleotides containing FapyG and FapyA at defined sites was recently developed (17Haraguchi K. Delaney M.O. Wiederholt C.J. Sambandam A. Hantosi Z. Greenberg M.M. J. Am. Chem. Soc. 2002; 124: 3263-3269Crossref PubMed Scopus (94) Google Scholar, 18Jiang Y.L. Wiederholt C.J. Patro J.N. Haraguchi K. Greenberg M.M. J. Org. Chem. 2005; 70: 141-149Crossref PubMed Scopus (32) Google Scholar). Here, we have reported the first study of the repair of FapyG and FapyA in these oligonucleotides, using nuclear and mitochondrial extracts of wild type and mice deficient in the two major glycosylases for the repair of oxidative DNA damage, OGG1 and NTH1. We determined the endogenous levels of FapyA, FapyG, and 8-oxoG in genomic DNA from wild type mice and found that FapyG levels are significantly higher than 8-oxoG. Our results showed that FapyG is repaired mainly by OGG1, both in nucleus and in mitochondria. NTH1 is the major glycosylase for the repair of FapyA. We found significantly higher levels of FapyG and FapyA in DNA from the knock-out mice, emphasizing the biological significance of OGG1 and NTH1 in the repair of these lesions. Materials—T4 polynucleotide kinase was from Stratagene (La Jolla, CA). [γ-32P]ATP (3000 Ci/mmol) was from PerkinElmer Life Sciences. G25 spin columns were from Amersham Biosciences. Nuclease P1 (from Penicillium citrinum) was from Calbiochem. Snake venom phosphodiesterase was obtained from Sigma. Alkaline phosphatase was purchased from Roche Diagnostics. 8-Hydroxy-2′-deoxyguanosine-15N5 (8-oxo-dG-15N5), 4,6-diamino-5-formamidopyrimidine-13C,15N2 (FapyA-13C,15N2), and 2,6-diamino-4-hydroxy-5-formamidopyrimidine-13C,15N2 (FapyG-13C,15N2) were purchased from Cambridge Isotope other were from Sigma. of and mice were by of and Cancer and and at the type were were by and the were and and mitochondrial extracts were as Free Radic. Biol. Med. PubMed Scopus Google Scholar). were by the and and in with Ref. of for the and of of of of the oligonucleotides used are in or oligonucleotides were as (17Haraguchi K. Delaney M.O. Wiederholt C.J. Sambandam A. Hantosi Z. Greenberg M.M. J. Am. Chem. Soc. 2002; 124: 3263-3269Crossref PubMed Scopus (94) Google Scholar, 18Jiang Y.L. Wiederholt C.J. Patro J.N. Haraguchi K. Greenberg M.M. J. Org. Chem. 2005; 70: 141-149Crossref PubMed Scopus (32) Google Scholar). The oligonucleotides were by and the the are in The oligonucleotide containing 8-oxoG was obtained from glycol substrate was generated as in Ref. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google oligonucleotides were using polynucleotide kinase and as in Ref. Res. PubMed Scopus Google FapyG, and oligonucleotides were to the in by the at for and to FapyA oligonucleotide was as that was at for used in are in The are in 8-oxoG in a DNA of FapyG was in a containing and of The were at for and with the of of and of followed by of at at the abasic was and at for equal of was and the were at for and by on a were by and using the The of incision was as the of in the product to the and of substrate of oligonucleotide was as that of substrate were of and oligonucleotides was using and of respectively. The were as with DNA were as for the glycosylase with the of at for the were by of and the were at for were in The were using of DNA from DNA was using a of the of were and in of containing and The were then in containing and and at for to cell of was and the were at for a in were by at for The was and the DNA was with of at The DNA was by in of and with at for The were then to in at for in and as DNA was from the with and in by and was used to and 8-oxoG as 8-hydroxy-2′-deoxyguanosine in DNA from and knock-out mice. of DNA were with of as an DNA were with venom and phosphatase for and then by as in Ref. P. J. Dizdaroglu M. Res. 2004; PubMed Scopus Google For and was used to the of and and and M. Jaruga P. H. Free Radic. Biol. Med. PubMed Scopus Google Scholar). The and of FapyG and FapyA in DNA was by by E. coli as in Ref. P. Jaruga P. T. H. Dizdaroglu M. 2004; 34: PubMed Scopus Google Scholar). of DNA were with of and of as and then with of DNA and were by were and by as P. Jaruga P. T. H. Dizdaroglu M. 2004; 34: PubMed Scopus Google Scholar). For and was used to the of FapyA and FapyG and M. 1993; PubMed Scopus Google Scholar). Endogenous of FapyG and FapyA in levels in cellular DNA have been determined in mice to T. Hori Y. Dizdaroglu M. Int. J. Radiat. Biol. 1993; 64: 645-650Crossref PubMed Scopus (62) Google Scholar). We determined background levels of FapyA, and FapyG in liver DNA from mice of 8-oxoG and FapyA were FapyG on the other were significantly higher than 8-oxoG and FapyA, at Jaruga P. Speina E. D. Tudek B. Olinski R. Res. 2000; 28: PubMed Scopus Google Scholar) also found FapyG levels to be higher than 8-oxoG levels in liver DNA. of FapyG in are the major cellular of and the mitochondrial DNA has been to be to higher oxidative damage than nuclear DNA Free Radic. Res. 1998; PubMed Scopus Google Scholar). We how FapyG and FapyA are repaired in mammalian using mouse liver mitochondrial extracts from and knock-out mice for OGG1 and the two major DNA glycosylases for oxidative base lesions. These extracts were free of nuclear as by an nuclear was used as a for mitochondrial extracts from mice the oligonucleotide in a and with the and of from mice showed incision at but incision with than of from mice significantly incision of the and extracts from mice showed at high DNA glycosylases that base intermediates with the which can be in the of B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). we the formation of in from and mice but not in from the knock-out We two in the extracts. OGG1 and NTH1 knock-out extracts showed one to the two in the The in with OGG1 These results that both OGG1 and NTH1 can intermediates with a However, the results in that OGG1 is the major DNA glycosylase for FapyG in mouse liver and that NTH1 a The residual incision with extracts from the knock-out mice that DNA glycosylase may a role in FapyG repair in liver mitochondria. We showed that OGG1 is the DNA glycosylase for removal of 8-oxoG from T. T. E. A. Cancer Res. Google Scholar). levels of FapyG in DNA in are higher than of 8-oxoG we the of base excision by for and substrates of we to of incision activity is as the of substrate of of product FapyG incision activity was higher than that of 8-oxoG. of both lesions was in extracts from OGG1 knock-out mice However, extracts FapyG that DNA glycosylase may in FapyG repair in the absence of of FapyA in the repair of FapyA lesions in mitochondrial extracts. oligonucleotides were by in a and incision activity was for extracts from mice. In and extracts showed a in incision activity with higher than was no in activity the two extracts. that NTH1 is the major glycosylase for FapyA removal in mitochondrial extracts and that OGG1 most not in In with the incision we in and but not in and extracts FapyA incision in and was with at that DNA glycosylase may for the absence of NTH1. However, we not any in extracts with and are repaired in mouse liver by NTH1 B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). we excision of FapyA and extracts the substrate with as that of a substrate containing In of a major role for NTH1 in the removal of these two lesions in liver mitochondria, incision activity for both lesions was in although incision activity was than FapyA incision of NEIL1 in that mitochondrial extracts from a excision activity or substrates suggested the of DNA glycosylase lesions. mammalian homologues have been and DNA (for and have been (14Hazra 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, T.K. Kow Y.W. Z. Imhoff B. Boldogh I. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar, S. DNA 2002; PubMed Scopus Google Scholar, I. T. M. E. Res. 2002; PubMed Scopus Google Scholar, M. S. K. S. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). The substrate of human and mouse NEIL1 indicate that FapyA and FapyG are substrates (14Hazra 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, 15Jaruga P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (80) Google Scholar). NEIL1 has been identified as the glycosylase for in nuclear extracts from mice M. S. K. S. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). NEIL1 is a for the residual incision activity we in from the mice. However, to date, of the glycosylases have been to mitochondria. We liver NEIL1 by with human NEIL1 as a NEIL1 was at in both and that glycosylase is in mouse and could account for the residual excision activity we in the absence of OGG1 and NTH1. of FapyG/FapyA in the excision of and substrates by liver nuclear extracts from the knock-out mice used for of mitochondria. FapyG was with by nuclear extracts from and and A reduction in incision was with from a major role for OGG1 in FapyG repair in the we not any in incision activity in and the in extracts from mice when with may to we in the mitochondrial NTH1 a role in FapyG removal in the nucleus in the absence of FapyA was also efficiently by but incision was in extracts from in activity was in that OGG1 not in the repair of FapyA in the as we in mitochondria. the other FapyA incision was in extracts than in a of one or DNA glycosylases in the FapyA is an substrate for both NTH1 and and a role for NTH1 in FapyA repair in NEIL1 could also contribute to the repair of FapyA in we FapyA incision in cell extracts from and of and knock-out mice the of these two glycosylases is to significantly among mouse tissues M. S. K. S. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). In NEIL1 is at a significantly higher level than in NTH1 is than and in levels for both glycosylases have been M. S. K. S. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar). FapyA incision by cell extracts from and from and knock-out mice showed a with the levels determined by M. S. K. S. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google incision activity was significantly in liver cell extracts lacking NTH1 (from and but in from these cell extracts from mice lacking NTH1 and of these results suggested that the of glycosylase for the repair of FapyA on levels. of DNA in DNA from from the results is that mice lacking OGG1 NTH1 Fapy lesions in DNA. We by levels of FapyG and of FapyA and 8-oxoG in liver genomic DNA from and mice The levels of these lesions in mice in are to with the knock-out mice. DNA from mice significantly higher levels of both FapyA and FapyG than the with that NTH1 is the major DNA glycosylase for FapyA and may also contribute to the removal of In DNA from OGG1 knock-out mice significantly higher levels of 8-oxoG and FapyG, but not FapyA, in with the incision DNA from the knock-out mice significantly higher levels of modified We have that FapyG in mouse liver DNA at higher levels than which is a biomarker of oxidative DNA damage. Fapy lesions have been to be miscoding of polymerases (9Wiederholt C.J. Greenberg M.M. J. Am. Chem. Soc. 2002; 124: 7278-7279Crossref PubMed Scopus (86) Google Scholar, B. M. Zastawny T.H. T. J. PubMed Scopus Google Scholar, B. J. Biol. 2003; PubMed Google Scholar), repair is for of both the nuclear and the mitochondrial The of the studies on the repair of formamidopyrimidines used a Me-FapyG analogue or DNA, which oxidatively induced DNA In we the repair of Fapy lesions by mitochondrial and nuclear extracts using oligonucleotides containing a single FapyG or FapyA. We have that both lesions are efficiently repaired in mouse liver and FapyG is with higher than 8-oxoG in mitochondrial extracts from mice, and FapyA is repaired with the as thymine glycol lesions. we have that FapyG, FapyA, and 8-oxoG in liver DNA from knock-out mice lacking OGG1 and underscoring the biological role of these glycosylases in the repair of is the first study the removal of formamidopyrimidines from DNA using mammalian cell extracts. We found that FapyG is by OGG1, both in and in In a NTH1 can also in FapyG opposite a base that could be formed by the misincorporation of opposite FapyG DNA purified E. coli endonuclease III both FapyA and FapyG from DNA, although FapyA was C.J. Patro J.N. Y.L. Haraguchi K. Greenberg M.M. Res. 2005; PubMed Scopus Google Scholar). The of NTH1 to FapyG repair was also by the of a in extracts from liver not in extracts from animals and by the elevated levels of FapyG in liver genomic DNA from mice. the other we not any that OGG1 can upon FapyA in mitochondrial or nuclear extracts. is in with the that purified human OGG1 not release FapyA lesions from DNA Dizdaroglu M. S. Res. PubMed Scopus Google Scholar). a of the incision activity FapyG and FapyA was in extracts from the knock-out mice lacking OGG1 and we residual incision both in mitochondrial and in nuclear that glycosylase could contribute to the incision The identified human and mouse NEIL1 efficiently FapyG and FapyA from DNA in vitro (14Hazra 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, 15Jaruga P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (80) Google Scholar), for residual It has been suggested that NEIL1 I. T. M. E. Res. 2002; PubMed Scopus Google Scholar) and T.K. Kow Y.W. Z. Imhoff B. Boldogh I. Mitra S. Izumi T. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) to the However, results have for the first that NEIL1 is also found in mouse liver and could account for the residual incision in the knock-out extracts. It is of to the of DNA glycosylase to the repair of one lesion in the enzymes are and for the We addressed with to FapyA incision using cell extracts from tissues with of NTH1 and Our results suggested that the level of to the repair NEIL1 (14Hazra 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) and NTH1 M. E. T. E. Mutat. Res. 2000; PubMed Scopus Google Scholar) levels in a role for both glycosylases in repair. However, are with NTH1 in and and NEIL1 in and it is that these two glycosylases biological in is that biological are on the of For OGG1 by kinase M. E. Res. 2002; PubMed Scopus Google Scholar), whereas by kinase J. K. Res. 2005; PubMed Scopus Google Scholar). In we found that mouse liver as suggested by (14Hazra 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) and M. S. K. S. A. J. Biol. Chem. 2002; 277: Full Text Full Text PDF PubMed Scopus Google Scholar) can as a activity for 8-oxoG and thymine glycol repair. However, we showed that in mouse liver mitochondria, 8-oxoG is repaired by OGG1 T. T. E. A. Cancer Res. Google Scholar) and that thymine glycol lesions are repaired by NTH1 B. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). We results in and we showed that in the absence of OGG1 or mitochondrial extracts can FapyG and FapyA, respectively. results suggested that NEIL1 may as a glycosylase for in mouse but not for 8-oxoG and studies to 8-oxoG excision by human (14Hazra 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, T.A. E. H. DNA 2003; PubMed Scopus Google Scholar) and mouse NEIL1 P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (80) Google Scholar) and showed a for the excision of in with FapyG/FapyA by mouse NEIL1 P. Birincioglu M. Rosenquist T.A. Dizdaroglu M. Biochemistry. 2004; 43: 15909-15914Crossref PubMed Scopus (80) Google Scholar). These results with purified enzymes that NEIL1 not in the repair of 8-oxoG and in mouse mitochondria. study has for the first that formamidopyrimidines are efficiently repaired in cellular extracts and that OGG1 and NTH1 major in The results have also indicated that NEIL1 may in base excision repair of in tissues in which it is expressed. Our results suggested that Fapy lesions are substrates of these enzymes and that these lesions may be DNA modifications to and We M. III and for the of with with