5′-Nicked Apurinic/Apyrimidinic Sites Are Resistant to β-Elimination by β-Polymerase and Are Persistent in Human Cultured Cells after Oxidative Stress

Genomic DNA is continuously exposed to oxidative stress. Whereas reactive oxygen species (ROS) preferentially react with bases in DNA, free radicals also abstract hydrogen atoms from deoxyribose, resulting in the formation of apurinic/apyrimidinic (AP) sites and strand breaks. We recently reported high steady-state levels of AP sites in rat tissues and human liver DNA (Nakamura, J., and Swenberg, J. A. (1999) Cancer Res. 59, 2522–2526). These AP sites were predominantly cleaved 5′ to the lesion. We hypothesized that these endogenous AP sites were derived from oxidative stress. In this investigation, AP sites induced by ROS were quantitated and characterized. A combination of H2O2 and FeSO4 induced significant numbers of AP sites in calf thymus DNA, which were predominantly cleaved 5′ to the AP sites (75% of total aldehydic AP sites). An increase in the number of 5′-AP sites was also detected in human cultured cells exposed to H2O2, and these 5′-AP sites were persistent during the post-exposure period. β-Elimination by DNA β-polymerase efficiently excised 5′-regular AP sites, but not 5′-AP sites, in DNA from cells exposed to H2O2. These results suggest that 5′-oxidized AP sites induced by ROS are not efficiently repaired by the mammalian short patch base excision repair pathway. Genomic DNA is continuously exposed to oxidative stress. Whereas reactive oxygen species (ROS) preferentially react with bases in DNA, free radicals also abstract hydrogen atoms from deoxyribose, resulting in the formation of apurinic/apyrimidinic (AP) sites and strand breaks. We recently reported high steady-state levels of AP sites in rat tissues and human liver DNA (Nakamura, J., and Swenberg, J. A. (1999) Cancer Res. 59, 2522–2526). These AP sites were predominantly cleaved 5′ to the lesion. We hypothesized that these endogenous AP sites were derived from oxidative stress. In this investigation, AP sites induced by ROS were quantitated and characterized. A combination of H2O2 and FeSO4 induced significant numbers of AP sites in calf thymus DNA, which were predominantly cleaved 5′ to the AP sites (75% of total aldehydic AP sites). An increase in the number of 5′-AP sites was also detected in human cultured cells exposed to H2O2, and these 5′-AP sites were persistent during the post-exposure period. β-Elimination by DNA β-polymerase efficiently excised 5′-regular AP sites, but not 5′-AP sites, in DNA from cells exposed to H2O2. These results suggest that 5′-oxidized AP sites induced by ROS are not efficiently repaired by the mammalian short patch base excision repair pathway. reactive oxygen species 8-hydroxy-2′-deoxyguanine apurinic/apyrimidinic aldehyde reactive probe slot-blot 2,2,6,6-tetramethylpiperidinoxyl methoxyamine E. coli exonuclease III E. coli endonuclease III high pressure liquid chromatography DNA β-polymerase deoxyribose phosphate Reactive oxygen species (ROS)1 are generated continuously in cells during normal metabolic processes and by a number of exogenous agents, including ionizing radiation. ROS can react with cellular components such as proteins, lipids, and nucleic acids to induce DNA adducts such as 8-hydroxy-2′-deoxyguanosine (8-OH-dG) (1.Beckman K.B. Ames B.N. J. Biol. Chem. 1997; 272: 19633-19636Abstract Full Text Full Text PDF PubMed Scopus (834) Google Scholar,2.Ames B.N. Gold L.S. Mutat. Res. 1991; 250: 3-16Crossref PubMed Scopus (676) Google Scholar). It is believed that these oxidized bases are predominantly repaired by a base excision repair pathway (3.Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar). In this process, a bifunctional 8-OH-dG-DNA glycosylase with apurinic/apyrimidinic (AP) lyase such as 8-hydroxy-2′-deoxyguanine-DNA glycosylase cleaves theN-glycosylic bond between 8-hydroxyguanine and deoxyribose and incises immediately 3′ to AP sites, leaving 3′-nicked AP sites (4.Bjoras M. Luna L. Johnsen B. Hoff E. Haug T. Rognes T. Seeberg E. EMBO J. 1997; 16: 6314-6322Crossref PubMed Scopus (330) Google Scholar,5.Rosenquist T.A. Zharkov D.O. Grollman A.P. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 7429-7434Crossref PubMed Scopus (456) Google Scholar). The 3′-AP sites generated by the DNA glycosylase are subsequently excised by class II AP endonuclease (3.Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar), resulting in a 3′-hydroxyl group and a 5′-phosphate group. Repair is completed by polymerase and ligase activity. Recently, it has been reported that mammalian cell extracts repair 8-OH-dG preferentially via single nucleotide replacement reactions (6.Dianov G. Bischoff C. Piotrowski J. Bohr V.A. J. Biol. Chem. 1998; 273: 33811-33816Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7.Fortini P. Parlanti E. Sidorkina O.M. Laval J. Dogliotti E. J. Biol. Chem. 1999; 274: 15230-15236Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). The contribution of nucleotide excision repair to the removal of 8-OH-dG was not significant in experiments using human cell extracts (6.Dianov G. Bischoff C. Piotrowski J. Bohr V.A. J. Biol. Chem. 1998; 273: 33811-33816Abstract Full Text Full Text PDF PubMed Scopus (202) Google Scholar, 7.Fortini P. Parlanti E. Sidorkina O.M. Laval J. Dogliotti E. J. Biol. Chem. 1999; 274: 15230-15236Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). These results indicate that base excision repair plays a central role in counteracting oxidized base lesions. In addition to base damage in DNA, ROS also induce lesions by hydrogen abstraction of the deoxyribose, frequently producing oxidized AP sites as well as DNA strand breaks (8.Breen A.P. Murphy J.A. Free Radical Biol. Med. 1995; 18: 1033-1077Crossref PubMed Scopus (910) Google Scholar). AP sites are also generated spontaneously by chemical depurination of labile oxidized bases and enzymatically by DNA glycosylases as mentioned above. Hydrogen abstraction has been examined extensively for model deoxyribose and polynucleotides (9.Von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis Ltd., London1987: 238-249Google Scholar). Although <10% of the hydroxyl radicals attack sugar residues in single-stranded polynucleotides, it has been proposed that oxidized AP sites induced by ROS may be one of the major oxidative lesions in double-stranded DNA (3.Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar, 9.Von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis Ltd., London1987: 238-249Google Scholar). These studies demonstrated that all hydrogen atoms of deoxyribose and ribose are potential targets for direct attack by oxygen radicals. In B-form duplex DNA, however, hydrogen atoms at the C-4′ and C-5′ positions of deoxyribose are the most accessible to ROS (10.Pogozelski W.K. Tullius T.D. Chem. Rev. 1998; 98: 1089-1108Crossref PubMed Scopus (977) Google Scholar). ROS-induced sugar lesions and strand cleavage in genomic DNA are difficult to examine, mainly due to the large variety of products as well as their instability even at mild temperatures and neutral pH (11.Lafleur M.V.M. Woldhuis J. Loman H. Int. J. Radiat. Biol. 1981; 39: 113-118Google Scholar). Many oxidized sugars are very labile, as terminal sugar lesions tend to be modified spontaneously during experimental procedures. We recently developed a sensitive aldehyde reactive probe slot-blot (ASB) assay to detect aldehydic AP sites in DNA, which can quantitate <1 AP site/106 nucleotides (12.Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225PubMed Google Scholar). Using this assay, we detected 50,000–200,000 AP sites in mammalian cells under normal physiological conditions (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). Large numbers of AP sites were detected in brain, heart, and colon DNAs, which appear to be continuously exposed to higher levels of oxidative stress. These endogenous AP sites were predominantly cleaved 5′ to the AP sites. Therefore, we hypothesized that oxidative stress directly induces 5′-nicked oxidized AP sites, which may contribute to a high steady-state level of AP sites in mammalian cells and tissues. To test this hypothesis, we have quantitated and characterized AP sites induced by ROS. We also have examined the repair efficiency of these AP sites in human cultured cells. Thymus was harvested from a newborn Holstein calf and quickly frozen on dry ice. After thawing, the calf thymus was homogenized in lysis buffer (Gentra Systems, Inc.) with 10 mm2,2,6,6-tetramethylpiperidinoxyl (TEMPO; Aldrich) on ice. DNA was then isolated by phenol/Sevag (chloroform:isoamyl alcohol, 24:1) extraction and purified as described (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). Calf thymus DNA was with 10 in 10 and purified as described (12.Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225PubMed Google Scholar). Calf thymus DNA isolated in calf thymus DNA with was with H2O2 FeSO4 in 10 at for 10 with The AP assay was immediately the of oxidative base the was by addition of and the DNA was by with After the DNA with DNA was in The AP assay was a modified from that reported by and Swenberg (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). of DNA in of was with aldehyde reactive probe at for 10 After using DNA was in buffer pH The DNA was by a and the DNA was then at of DNA was on a The was with and then in a for The was with 10 of for and then in the at for After the the on the was by The was then exposed to and the developed was using The AP cleavage assay was as described (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google with a The number of total AP sites was by the assay as described above. of DNA and coli exonuclease III Inc.) were in of 10 and at for and immediately by the of DNA, 10 and were in of 10 at for and immediately by the of DNA and of exonuclease III in of 10 were at for immediately by addition of of The was with in the buffer at for immediately by the bases are repaired by E. III leaving AP sites on the DNA (3.Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar). III was by Kow The assay was as described (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). of 8-OH-dG was on that was modified from a described by C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). DNA was enzymatically to using and The was by and 8-OH-dG was quantitated using was at and The of 8-OH-dG was to the of DNA as by cells were as cells from the Cancer at the of at After cells were in of modified Inc.) The cultured cells were exposed to H2O2 at for immediately by After with cell were frozen and at To test the repair efficiency of oxidative DNA cells in were in of modified with and cultured at for to DNA from cultured cells was using the DNA extraction (Gentra Systems, cell were and in lysis buffer with After with a the in the was with The was in lysis buffer with 10 and with and A at for by and DNA The DNA was in with The DNA was at for The AP repair assay was by a modified from the AP cleavage of DNA with buffer (12.Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225PubMed Google and of III were in of and at for to 5′-nicked AP sites. In this was of to DNA by the exonuclease of The DNA was subsequently with human from S. H. of at in of and at for The aldehyde reactive probe was in the with of of and of 10 aldehyde reactive probe at for 10 and immediately by the of DNA to the was with human at as described and by the AP The efficiency of AP repair was by the of AP sites by by the of AP sites by of the most significant oxygen radicals is the hydroxyl which is generated by the of with H2O2 via the P. S. J. Biol. Chem. 1999; 274: Full Text Full Text PDF PubMed Scopus Google Scholar). To oxygen radicals induced by the directly AP sites in DNA, calf thymus DNA with was with 10 FeSO4 with H2O2 at for 10 under neutral pH The number of AP sites in calf thymus DNA with 10 and was by H2O2 a a group (9.Von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis Ltd., London1987: 238-249Google Scholar), is to the number of in mammalian tissues at T. L. Chem. Res. 1998; PubMed Scopus Google Scholar). To AP formation by the calf thymus DNA was with 10 H2O2 and AP formation in a and DNA from AP formation at of 10 To test AP sites are major oxidative lesions induced by the we the number of AP sites, sites, and in calf thymus DNA the III cleaves theN-glycosylic bond between deoxyribose and most oxidized leaving AP sites (3.Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar). The number of sites was from the number of AP sites with III the number of AP sites with calf thymus DNA large of oxidative base lesions even we isolated DNA from calf thymus with 10 in this Whereas the steady-state level of AP sites was detected at nucleotides in isolated calf thymus DNA, endogenous 8-OH-dG was not and sites were the Using calf thymus DNA isolated in this a combination of 10 and 10 sites and 8-OH-dG at and These results that the induced by 10 H2O2 and 10 FeSO4 predominantly by base lesions and AP sites of sites, and AP sites was In addition to AP sites, the formation of these oxidative base lesions by the was by at from 10 to and In the we isolated DNA from cultured cells with lysis buffer with to formation of oxidative base lesions as well as AP sites. ROS can induce sugar lesions directly by hydrogen abstraction of deoxyribose, resulting in AP sites as well as DNA strand breaks. AP sites are also generated spontaneously by chemical depurination of labile oxidized bases and bases and enzymatically by cleavage of the bond between the sugar and modified We recently developed AP cleavage assay to the of cleavage at AP sites (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). To test the AP sites induced by ROS were 3′-nicked the AP cleavage assay was for calf thymus DNA exposed to the calf thymus DNA AP was with 10 and 10 10 The number of AP sites to AP nucleotides In this assay, we III as the class II AP endonuclease to of AP sites and to detect the DNA was with III by the A single of III the number of AP sites to AP was to the we (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google and may be due to the combination of on the by III and of AP sites during with In in significant of the number of AP sites. After with III by the number of AP sites was by from the number of AP sites in calf thymus DNA exposed to the The of and cleaved AP sites and aldehydic lesions are in A major was that the AP cleavage induced by the reactions were from induced by depurination (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). To AP formation in cellular DNA by oxygen we exposed cells to H2O2 at at for of H2O2 to cells was by the The of cells was the cultured cells were cells a increase in the number of AP sites to H2O2 in a The number of sites and was also by with H2O2 of of sites, and AP sites at 10 was and we that H2O2 induces the in cellular DNA, AP sites one of the major oxidative DNA lesions in cells. these suggest that the repair of 8-OH-dG may be with the repair of AP sites and oxidized base of in cells exposed to 10 nucleotides was from to numbers of were not for The were for the as and 8-OH-dG was from to The numbers of were not for The were for the as and in a To the repair efficiency of these oxidative DNA the cultured cells were in with for to the to 10 H2O2. 8-OH-dG was repaired and oxidized were repaired In we detected in the number of AP sites the repair period. The that AP sites induced by H2O2 are to cellular excision repair with oxidized The AP sites in cells exposed to H2O2 were characterized using the AP cleavage The number of 5′-AP sites and aldehydic lesions with to 10 H2O2 These lesions to during the repair period. In the of 3′-nicked and AP sites not increase in cells exposed to H2O2. To the of 5′-AP sites in cells to H2O2, we 5′-AP sites by oxidative stress. calf thymus DNA exposed to the to buffer by the 5′ to AP sites by III was with The efficiency of AP repair was by the of AP sites by by the of AP sites by efficiently excised 5′-regular AP sites at a of In 5′-AP sites directly by ROS were efficiently excised from the DNA by To 5′-AP sites in cells exposed to H2O2 are repaired 5′-regular AP sites 5′-AP the DNA from cells exposed to was with at by the These 5′-AP sites were also excised efficiently by with 5′-regular AP sites. We also detected a AP a of III and not repair assay by The efficiency of excision of 5′-nicked AP sites by the lyase of was for 5′-regular AP sites and 5′-AP in calf thymus DNA and for 5′-AP sites in DNA from cells immediately to H2O2. The efficiency of AP repair was by the of AP sites by by the of AP sites by The were from of Large A large number of AP sites are continuously by depurination in mammalian cells (12.Nakamura J. Walker V.E. Upton P.B. Chiang S.-Y. Kow Y.W. Swenberg J.A. Cancer Res. 1998; 58: 222-225PubMed Google Scholar), leaving AP sites. stress also induces labile adducts that in AP sites by chemical These AP sites are subsequently 5′ to AP sites by class II AP oxidative base lesions are also excised by bifunctional DNA glycosylases with AP lyase which 3′-AP sites. In hydrogen abstraction directly induces and 3′-nicked AP sites (8.Breen A.P. Murphy J.A. Free Radical Biol. Med. 1995; 18: 1033-1077Crossref PubMed Scopus (910) Google Scholar, 9.Von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis Ltd., London1987: 238-249Google Scholar, W.K. Tullius T.D. Chem. Rev. 1998; 98: 1089-1108Crossref PubMed Scopus (977) Google Scholar). Therefore, a significant number of and AP sites may be induced in cells under oxidative stress The demonstrated that oxidative stress predominantly induced AP sites in and in 5′-nicked AP sites directly induced by ROS were efficiently from the DNA by but not by In AP sites induced by by III were efficiently excised by These results indicate that the 5′-AP sites induced in and in by ROS are repaired 5′-regular AP sites. In B-form duplex DNA, ROS most induce sugar lesions directly by abstraction of hydrogen atoms at the C-4′ C-5′ of deoxyribose (9.Von Sonntag C. The Chemical Basis of Radiation Biology. Taylor & Francis Ltd., London1987: 238-249Google Scholar, W.K. Tullius T.D. Chem. Rev. 1998; 98: 1089-1108Crossref PubMed Scopus (977) Google Scholar, M.V.M. Woldhuis J. Loman H. Int. J. Radiat. Biol. 1981; 39: 113-118Google Scholar). hydrogen abstraction at C-4′ results in DNA cleavage to the the base and the phosphate In under hydroxyl radicals induce sites with between aldehydic AP sites. Hydrogen abstraction at C-5′ has also been proposed to aldehydic AP sites under conditions (8.Breen A.P. Murphy J.A. Free Radical Biol. Med. 1995; 18: 1033-1077Crossref PubMed Scopus (910) Google Scholar, M.V.M. Woldhuis J. Loman H. Int. J. Radiat. Biol. 1981; 39: 113-118Google Scholar). These AP sites with aldehydic be a for and are by the Therefore, we that the 5′-AP sites induced by ROS oxidized AP sites such as AP sites. Whereas excised 5′-regular AP sites at efficiently cleaved 3′ to the 5′-AP sites at These results are in with the in the efficiency of cleavage of and 5′-nicked AP sites by T. A. PubMed Scopus Google and J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). A duplex DNA with a single AP was cleaved by with and for at J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). In we genomic DNA a of AP sites which to be a on the number of endogenous AP sites (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). excised the in genomic DNA as efficiently as in at These suggest that efficiently and AP sites under AP sites were oxidative DNA lesions generated by the in in these AP sites one of the major oxidative lesions in genomic DNA from cells exposed to H2O2. AP sites were persistent with oxidative base lesions in cultured cells to oxidative stress. described but not efficiently excised 5′-AP sites induced by ROS. These indicate that 5′-AP sites induced by oxidative stress are not repaired efficiently by cellular excision repair the results the efficiency of repair by indicate that the lesions are by It has been proposed that the in a base with the AP and cleaves 3′ to the AP J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar). The in lyase between and for 5′-AP sites induced by ROS that the of in may not efficiently the aldehydic of 5′-nicked oxidized AP sites. be as these AP sites induced by 5′-oxidized AP sites, in not the aldehydic of these AP sites due to of oxidized AP sites. Although 5′-nicked AP sites induced by by human AP endonuclease are excised by B. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar), the excision of for 5′-nicked AP sites directly induced by oxidative stress are We that 5′-oxidized AP sites directly induced by ROS may be repaired by the patch base excision pathway. In we large numbers of endogenous 5′-nicked AP sites in rat tissues and human liver (13.Nakamura J. Swenberg J.A. Cancer Res. 1999; 59: 2522-2526PubMed Google Scholar). the cleavage of AP sites induced by the are to of endogenous AP sites in rat and human tissues. Although it has been believed that AP sites are efficiently oxidized AP sites are not excised as efficiently as AP sites in cells. Therefore, we that endogenous AP sites from oxidized AP sites from AP sites. We suggest that the high steady-state level of AP sites be due to short patch base excision repair pathway by It was demonstrated that lyase 1995; PubMed Scopus Google Scholar). J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google proposed that by is on of lyase by and of lyase by with results also that of efficiently excised from the DNA These indicate that in not for the lyase of The human counteracting most oxidative base lesions are bifunctional DNA glycosylases such as human 8-hydroxy-2′-deoxyguanine-DNA glycosylase and human endonuclease III T. C. J. T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar), leaving 3′-AP sites modified class II AP endonuclease the by to a group for DNA repair (3.Demple B. Harrison L. Annu. Rev. Biochem. 1994; 63: 915-948Crossref PubMed Scopus (1282) Google Scholar). Although ROS induce significant numbers of oxidized base was of the of and 3′-nicked AP sites in cellular DNA to H2O2. These suggest that class II AP endonuclease efficiently a large number of AP sites. In in repair 8-OH-dG and oxidized were repaired mainly by a short patch base excision repair pathway (6.Dianov G. Bischoff C. Piotrowski J. Bohr V.A. J. Biol. Chem. 1998; 273: 33811-33816Abstract Full Text Full Text PDF PubMed Scopus (202) Google P. Parlanti E. Sidorkina O.M. Laval J. Dogliotti E. J. Biol. Chem. 1999; 274: 15230-15236Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar, A. M. A. T. 1999; Full Text Full Text PDF PubMed Scopus Google Scholar). the DNA repair at 8-OH-dG was that at AP sites P. Parlanti E. Sidorkina O.M. Laval J. Dogliotti E. J. Biol. Chem. 1999; 274: 15230-15236Abstract Full Text Full Text PDF PubMed Scopus (193) Google Scholar). Therefore, it has been proposed that the processes from base to excision of 3′-AP sites may be suggest that to repair 3′-AP sites is not in base excision on these the excision of modified bases may be one of the processes in the 8-OH-dG base excision repair pathway. in human cultured oxidative stress induced AP endonuclease and cells to oxidative stress T. S. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). These results also the that 3′-AP sites generated by bifunctional DNA glycosylases may induce AP In addition to 3′-AP sites, ROS also induced including These be one of the for AP endonuclease in cells under oxidative stress Although the directly induced a significant number of AP sites in the in the number of AP sites was not in cells exposed to H2O2. AP sites and AP sites strand breaks directly induced by are repaired by of AP endonuclease and using B. J. Biol. Chem. 1998; 273: Full Text Full Text PDF PubMed Scopus Google Scholar, B. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). on these and the and oxidized aldehydic AP sites with cleavage on induced by ROS appear to be efficiently repaired in cells a base excision repair pathway. A high of the formation of AP sites, sites, and 8-OH-dG induced by a high level of oxidative stress. DNA from calf thymus also very of 8-OH-dG In the of steady-state levels of 8-OH-dG by has from to nucleotides in mammalian cells and tissues K.B. P.B. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus Google Scholar). are that induce oxidative DNA lesions during DNA extraction J. T. T. S. Mutat. Res. 1999; PubMed Scopus Google Scholar). The of free radicals by to be for DNA damage from oxidative stress. Therefore, the DNA extraction using a high of the of oxidative lesions during DNA In the 10 the number of by a of the These indicate that of oxidative DNA lesions also to as well as the repair of these lesions oxidative stress. studies are to the of 5′-AP sites in cells under normal physiological conditions as well as oxidative stress. Although H2O2 cells at a number of cells to 10 H2O2 and not These results suggest that 5′-oxidized AP sites are by cellular DNA repair Recently, Proc. Natl. Acad. Sci. U. S. A. 1998; PubMed Scopus (193) Google demonstrated that oxidative but not induces in proposed that a such as a strand is to contribute to genomic instability the of a It is that 5′-oxidized AP sites be in the in To it has been believed that AP sites are repaired very efficiently in genomic however, the high steady-state level of 5′-nicked AP sites as well as persistent AP sites oxidative stress suggest that of AP sites may not be efficiently repaired by the mammalian excision repair pathway. We S. H. and for human E. and calf

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