DNA-Protein Cross-link Formation Mediated by Oxanine

Chronic inflammation is a risk factor for many human cancers, and nitric oxide (NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. In this report, we report a novel genotoxic mechanism of NO that involves DNA-protein cross-links (DPCs) induced by oxanine (Oxa), a major NO-induced guanine lesion. When a duplex DNA containing Oxa at the site-specific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between Oxa and protein. The rate of DPC formation with DNA glycosylases was approximately two orders of magnitude higher than that with histone and HMG protein. Analysis of the reactivity of individual amino acids to Oxa suggested that DPC formation occurred between Oxa and side chains of lysine or arginine in the protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing Oxa. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis. Chronic inflammation is a risk factor for many human cancers, and nitric oxide (NO) produced in inflamed tissues has been proposed to cause DNA damage via nitrosation or oxidation of base moieties. Thus, NO-induced DNA damage could be relevant to carcinogenesis associated with chronic inflammation. In this report, we report a novel genotoxic mechanism of NO that involves DNA-protein cross-links (DPCs) induced by oxanine (Oxa), a major NO-induced guanine lesion. When a duplex DNA containing Oxa at the site-specific position was incubated with DNA-binding proteins such as histone, high mobility group (HMG) protein, and DNA glycosylases, DPCs were formed between Oxa and protein. The rate of DPC formation with DNA glycosylases was approximately two orders of magnitude higher than that with histone and HMG protein. Analysis of the reactivity of individual amino acids to Oxa suggested that DPC formation occurred between Oxa and side chains of lysine or arginine in the protein. A HeLa cell extract also gave rise to two major DPCs when incubated with DNA-containing Oxa. These results reveal a dual aspect of Oxa as causal damage of DPC formation and as a suicide substrate of DNA repair enzymes, both of which could pose a threat to the genetic and structural integrity of DNA, hence potentially leading to carcinogenesis. Nitric oxide (NO) 1The abbreviations used are: NO, nitric oxide; Oxa, oxanine; 8oxoG, 7,8-dihydro-8-oxoguanine; Xan, xanthine; Hx, hypoxanthine; dOTP, 2′-deoxyoxanosine 5′-triphosphate; DPC, DNA-protein cross-link; DPC1 and DPC2, major DPCs formed with HeLa cell extracts; HMG, high mobility group; Endo, endonuclease; Exo, exonuclease; Fpg, formamidopyrimidine glycosylase; AlkA, 3-methyladenine glycosylase II; mMPG, mouse methylpurine glycosylase; hNTH1, a human Endo III homolog; hOGG1, human 8-oxoguanine glycosylase; CBB, Coomassie Brilliant Blue; Pol, polymerase; DTT, dithiothreitol; PMSF, phenylmethylsulfonyl fluoride. synthesized from l-arginine by three isoforms of NO synthase mediates a variety of regulatory functions in vivo (1Marletta M.A. Cell. 1994; 78: 927-930Google Scholar, 2Wang Y. Marsden P.A. Adv. Pharmacol. 1995; 34: 71-90Google Scholar). A high flux of NO is produced by macrophages expressing inducible NO synthase during inflammatory responses (3MacMicking J. Xie Q.W. Nathan C. Annu. Rev. Immunol. 1997; 15: 323-350Google Scholar). Because chronic inflammation has been long recognized as a risk factor for many human cancers, it has been postulated that NO is carcinogenic by virtue of its ability to induce DNA damage (4Ohshima H. Bartsch H. Mutat. Res. 1994; 305: 253-264Google Scholar, 5Tamir S. Tannenbaum S.R. Biochim. Biophys. Acta. 1996; 1288: 31-36Google Scholar, 6Wink D.A. Vodovotz Y. Laval J. Laval F. Dewhirst M.W. Mitchell J.B. Carcinogenesis. 1998; 19: 711-721Google Scholar, 7Felley-Bosco E. Cancer Metastasis Rev. 1998; 17: 25-37Google Scholar). Although NO itself is not reactive to DNA, this molecule is converted to reactive nitrogen oxide species such as peroxynitrite (ONOO–) and nitrous anhydride (N2O3). ONOO– is a powerful oxidizing agent formed by the reaction with the superoxide anion ( O2·¯) that is simultaneously excreted from macrophages. ONOO– primarily oxidizes guanine to 7,8-dihydro-8-oxoguanine (8oxoG) (8Kennedy L.J. Moore Jr., K. Caulfield J.L. Tannenbaum S.R. Dedon P.C. Chem. Res. Toxicol. 1997; 10: 386-392Google Scholar), further degradation products of 8oxoG (9Tretyakova N.Y. Niles J.C. Burney S. Wishnok J.S. Tannenbaum S.R. Chem. Res. Toxicol. 1999; 12: 459-466Google Scholar), and 8-nitroguanine (10Yermilov V. Rubio J. Ohshima H. FEBS Lett. 1995; 376: 207-210Google Scholar, 11Yermilov V. Rubio J. Becchi M. Friesen M.D. Pignatelli B. Ohshima H. Carcinogenesis. 1995; 16: 2045-2050Google Scholar). 8oxoG is a potent mutagenic lesion inducing GC → TA transversions, whereas 8-nitroguanine readily undergoes depurination to yield an abasic site, a potent lethal as well as mutagenic lesion. N2O3 is a powerful nitrosating agent that is formed by autoxidation of NO in the presence of oxygen. Nitrosation of the primary amino group of guanine, adenine, and cytosine by N2O3 induces deamination, resulting in xanthine (Xan), hypoxanthine (Hx), and uracil (U), respectively (12Wink D.A. Kasprzak K.S. Maragos C.M. Elespuru R.K. Misra M. Dunams T.M. Cebula T.A. Koch W.H. Andrews A.W. Allen J.S. Keefer L.K. Science. 1991; 254: 1001-1003Google Scholar, 13Caulfield J.L. Wishnok J.S. Tannenbaum S.R. J. Biol. Chem. 1998; 273: 12689-12695Google Scholar). Nitrous acid (HNO2) also induces deamination of guanine, adenine, and cytosine by a similar mechanism (14Hartman Z. Henrikson E.N. Hartman P.E. Cebula T.A. Environ. Mol. Mutagen. 1994; 24: 168-175Google Scholar). We have previously identified a novel reaction product, oxanine (Oxa), that is formed by the nitrosation of guanine with NO or nitrous acid (15Suzuki T. Yamaoka R. Nishi M. Ide H. Makino K. J. Am. Chem. Soc. 1996; 118: 2515-2516Google Scholar, 16Suzuki T. Ide H. Yamada M. Endo N. Kanaori K. Tajima K. Morii T. Makino K. Nucleic Acids Res. 2000; 28: 544-551Google Scholar). Oxa and Xan were formed at a molar ratio of 1:3 when 2′-deoxyguanosine or DNA was treated with NO or nitrous acid. Oxa is also formed by incubation with N-nitrosoindoles (17Lucas L.T. Gatehouse D. Shuker D.E. J. Biol. Chem. 1999; 274: 18319-18326Google Scholar, 18Lucas L.T. Gatehouse D. Jones G.D. Shuker D.E. Chem. Res. Toxicol. 2001; 14: 158-164Google Scholar), showing that N-nitroso compounds produced by nitrosation of secondary amines by NO or nitrous acid can mediate Oxa formation. Although Oxa can be mutagenic and cytotoxic by directly affecting DNA replication or the stability of duplex DNA (19Suzuki T. Yoshida M. Yamada M. Ide H. Kobayashi M. Kanaori K. Tajima K. Makino K. Biochemistry. 1998; 37: 11592-11598Google Scholar, 20Suzuki T. Matsumura Y. Ide H. Kanaori K. Tajima K. Makino K. Biochemistry. 1997; 36: 8013-8019Google Scholar), there is another possibility: the O-acylisourea structure of Oxa is fairly reactive and can form an adduct or a DNA-protein cross-link (DPC) with nucleophilic cellular molecules (Fig. 1A). Indeed, we have recently shown in a model reaction that an Oxa-glycine adduct was formed when 2′-deoxyoxanosine and concentrated glycine were incubated in aqueous solution (21Suzuki T. Yamada M. Ide H. Kanaori K. Tajima K. Morii T. Makino K. Chem. Res. Toxicol. 2000; 13: 227-230Google Scholar). Similarly, a diazoate derivative of 2′-deoxycytidine, a reaction intermediate formed by NO or nitrous acid (22Suzuki T. Nakamura T. Yamada M. Ide H. Kanaori K. Tajima K. Morii T. Makino K. Biochemistry. 1999; 38: 7151-7158Google Scholar), also reacts with lysine and its homopolymer to yield T. Yamada M. Nakamura T. Ide H. Kanaori K. Tajima K. Morii T. Makino K. Chem. Res. Toxicol. 2000; 13: (Fig. the results from that the DNA repair for the mutagenic or cytotoxic of NO and nitrous acid is not base repair or repair (14Hartman Z. Henrikson E.N. Hartman P.E. Cebula T.A. Environ. Mol. Mutagen. 1994; 24: 168-175Google Scholar, Mutat. Res. 2000; Scholar, S. Burney S. Tannenbaum S.R. Chem. Res. Toxicol. 1996; Scholar, Tannenbaum S.R. J. 2001; Scholar, Hartman P.E. J. Scholar, M. Laval J. 1997; 12: Scholar). the of DPCs and the potentially formed by Oxa and cytosine it is that by or repair in DNA and D. Scholar, C. 1999; Scholar). with this it has been shown recently that DPC containing a adduct with a Y. S. Scholar). Thus, DPCs and induced by NO or nitrous acid mutagenic or cytotoxic in the of repair (14Hartman Z. Henrikson E.N. Hartman P.E. Cebula T.A. Environ. Mol. Mutagen. 1994; 24: 168-175Google Scholar, Mutat. Res. 2000; Scholar, S. Burney S. Tannenbaum S.R. Chem. Res. Toxicol. 1996; Scholar, Tannenbaum S.R. J. 2001; Scholar, Hartman P.E. J. Scholar, M. Laval J. 1997; 12: Scholar). for the model (21Suzuki T. Yamada M. Ide H. Kanaori K. Tajima K. Morii T. Makino K. Chem. Res. Toxicol. 2000; 13: 227-230Google Scholar, T. Nakamura T. Yamada M. Ide H. Kanaori K. Tajima K. Morii T. Makino K. Biochemistry. 1999; 38: 7151-7158Google Scholar), have been to DPC or formed between Oxa or cytosine diazoate in DNA and cellular In the we a duplex containing site-specific Oxa and the reactivity to proteins and We report that Oxa in DNA reacts with proteins such as histone, high mobility group (HMG) protein, and of DNA repair DNA as well as to form DPC formation is also in the incubation with the HeLa cell The of DPC formation with histone and DNA glycosylases two of DPC formation. and was synthesized and as previously (19Suzuki T. Yoshida M. Yamada M. Ide H. Kobayashi M. Kanaori K. Tajima K. Makino K. Biochemistry. 1998; 37: 11592-11598Google Scholar). with were synthesized by the A containing 8oxoG was with and to the as previously K. Yamada T. H. Y. Y. T. S. H. T. Ide H. J. Biol. Chem. 2000; Scholar). The of was and the duplex was and were from and HMG of and was from E. DNA in was from DNA glycosylases, E. III K. H. H. T. H. Y. S. Ide H. Biochemistry. 2000; Scholar), Endo K. Yamada T. Y. H. Y. S. Ide H. J. Biol. Chem. 2000; Scholar), formamidopyrimidine glycosylase K. Yamada T. H. Y. Y. T. S. H. T. Ide H. J. Biol. Chem. 2000; Scholar), and 3-methyladenine glycosylase H. Kobayashi M. Y. Ide H. J. Biol. Chem. 1999; 274: Scholar, H. Kobayashi M. S. Y. Yoshida M. Ide H. J. Biol. Chem. 1999; 274: Scholar), mouse methylpurine glycosylase R. T. T. S. Biochemistry. 1998; 37: Scholar), a human Endo III S. T. R. T. S. S. J. Biol. Chem. 1998; 273: and human 8-oxoguanine glycosylase K. Yamada T. H. Y. Y. T. S. H. T. Ide H. J. Biol. Chem. 2000; Scholar, Y. T. M. E. H. S. Biophys. Res. 1999; were as and were from the of an was an by a DNA reaction A was with and to a the position was The was incubated with and in and at for and were to the reaction and incubation was further at for DNA was by and a a of Oxa was the the in the was by of the reaction in a The resulting containing Oxa was to a by of and is not during the and not and (19Suzuki T. Yoshida M. Yamada M. Ide H. Kobayashi M. Kanaori K. Tajima K. Makino K. Biochemistry. 1998; 37: 11592-11598Google Scholar). The containing Oxa and the duplex were and containing in of Oxa at the position was to the and the resulting duplex was of with was incubated with of a histone or a of of or of HMG in at for to The was with an of and and by were also side by and The was with Coomassie Brilliant and to The of the was a The with DNA repair were in a similar The used were DNA glycosylases from human and mouse and E. Endo Fpg, and was incubated with of a DNA glycosylase in an at for to The of the incubation was and for and and for and for Endo Endo and and and for were by as for When the reaction was with proteins AlkA, Endo and histone were at for to The of hOGG1, AlkA, and histone not in in as from the of Endo was to of that to of the protein. Thus, the of Endo was in the reaction with Endo also for and of and Fpg, or Endo were incubated as an of the reaction was further treated with or in a by the at for to of the in the were by with HeLa cell extract was from HeLa were at or The were in of phenylmethylsulfonyl and DTT, and were by at for The cell was in of PMSF, and The were with a The was for and the was The containing cell was with a of PMSF, DTT, and by a of PMSF, DTT, and and for The was at for and the was PMSF, and for The extract was as at The was with a was incubated with the cell extract as in and at for The products were as for The reaction with the cell extract for was in a similar the with cell the cell extract as was incubated with or at for The a was a from of The cell extract with the was incubated with as and DPC was by In the cell extract as was incubated with containing 8oxoG in and at The was with at for to abasic and with acid. were with a and and by containing The of the was as for of with Acids and was incubated with an amino acid or an amino acid in and at for to DNA was by The was with and by The of the was as The with and and and were in a similar DPC with and HMG was incubated with individual histone ratio of for and the products were by the for the of histone and with to form DPCs that than DNA were in (Fig. The mobility of DPCs was than that of the proteins as well as DNA (Fig. The of and DPCs were from the mobility of and that of DPCs (Fig. The in the between histone and the DPCs were which to the of Thus, DPC a histone and at a ratio of of the DPC was not formed when the reaction was with containing in of Oxa at the not that the cross-link reaction was for Oxa. Because the of and and as respectively (Fig. DPCs to the also as in the (Fig. The rate of cross-link formation of Oxa was from the yield of DPC and the of histone in by reaction The in for the histone was at for and for was also incubated with a of histone similar formed DPC (Fig. the of DPC formed with the histone was than the of DPC formed from individual with at the and form a and and form a in the of DNA Science. Scholar, Science. Scholar). is that the between histone have reactive in the proteins or the of for the HMG also with to form DPC (Fig. The reaction of HMG were similar to for histone ratio of and incubation The DPC HMG and at a ratio of as from the in the (Fig. The of with HMG was to of histone of of proteins and the histone or HMG were incubated with and products were by as shown in The of proteins and DPCs were from the and The of and DPCs the with and histone, HMG protein, and DPCs formed with by and the of DNA glycosylases and DPCs the with DNA glycosylases and DPCs formed with by and rate of cross-link formation of oxanine with proteins and and amino acid of proteins were from the HMG Endo and Because there is report histone the for human histone were used for The for HMG were the and amino acid of and histone as the were as yield of cross-link The were from the at and for and and for DNA and for amino and and for of to that of of and The and amino acid of proteins were from the HMG Endo and Because there is report histone the for human histone were used for The for HMG were the and amino acid of Although and histone as the were as The yield of cross-link The were from the at and for and and for DNA and for amino and and for of to that of The of and in a DPC with DNA was incubated with DNA glycosylases from E. Endo Fpg, and and hOGG1, and for to The molar ratio of glycosylase was in the histone and HMG protein, Endo Fpg, AlkA, and formed DPCs (Fig. The DPC was of In Endo hNTH1, and not form DPC of the enzymes, the in the of DPC to was (Fig. that DPC was a of and Endo DPC species were the major DPC in the Because the Endo used a in by the DPCs be to Endo a for not which when a of Endo was used as in this The for the DNA glycosylases Fpg, AlkA, and were two orders of magnitude than for the histone and HMG of DPC formation for the DNA glycosylases and When the DNA glycosylases AlkA, and were by for to the DPC was not formed of incubation (Fig. similar of histone not DPC formation (Fig. These results that is for the DPC formation of DNA glycosylases, whereas is not for the DPC formation of with a report Laval J. Biophys. Res. 2001; Scholar), to when treated with Thus, the reaction with was not of the ability of DNA glycosylases and by and by DNA glycosylases AlkA, and or histone of were incubated with containing Oxa was with at for or were by of the and the of DNA and products of DPCs to and the of DPC as a DNA-protein the reaction incubation of with Fpg, or Endo was treated with or results of by The DPC formed with histone was by and (Fig. of the associated protein. the DPC was to with that has in histone and amino acids and and amino (Fig. Similarly, the DPC was by and (Fig. showing of the associated The DNA was also by the The DPCs containing and Endo were also to with and not to and and not These results that DPCs were of DNA and protein. Analysis of DPCs by from HeLa were incubated with at for of the products two major DPCs as DPC1 and (Fig. The formation of DPC1 and was as by of DPC1 and were formed in the presence of and with in not The of DPC1 and were and that the of proteins were and The mobility of DPC1 was than that of DPC formed between and (Fig. for DNA glycosylases (Fig. the formation of DPC1 and was to that DPC formation was when the cell extract was treated at for to the reaction (Fig. When the cell extract was incubated with DPC formation was not not Thus, and duplex DNA were for DPC formation with the cell The of a in DPC1 was which was than used as a has of which and than H. Y. T. R. C. T. M. K. M. Cancer Res. 1997; Scholar, K. T. H. T. D. K. Y. Mol. Biol. Cell. 1999; 10: Scholar). Thus, a of could rise to this was the the cell extract was treated with to When containing 8oxoG was incubated with the cell for in the the of was (Fig. and The of the was the of the In of cellular by the the formation of DPC1 (Fig. A and that was not in the formation of the of the of Acids and to the amino acid in DPC formation in amino acids were incubated with at for ratio of acid and products were by and formed with Oxa in (Fig. containing the amino acid adduct than in The adduct formation occurred with not containing in of Oxa at the position (Fig. Because the group of and is not in the for the reaction to the reaction with of the amino acids were further that the of and not of and with not These results that the side chains of and in in DPC formation. The reactivity of the side chains of and for was when of and (Fig. of the reactivity of and to Oxa. was incubated with and and and and at for the The of the amino acids and were The of Oxa in was by and is incubation Because a of amino acids formed with the reaction with and relevant was The were and and were and The ratio of for the reaction was the as that for amino acids ratio of and with to form (Fig. with of the converted to the adduct of incubation (Fig. The of and were higher than of of and Although also formed an the reactivity for was to of of and not The and not form of the reactivity of the amines for Oxa and to be the of amino in the molecule not the of this was also the for amino and a side with Oxa amino acids not the is to that between the DNA and the in the or amino acid side chains the of DNA and nucleophilic molecules to form NO and nitrous acid induce deamination of and resulting in and Hx, genotoxic of the and from DNA by glycosylase and both in and M. H. B. F. S. C. M. P.A. Nucleic Acids Res. Mol. Biol. 2001; Scholar, M. Laval J. S. 1994; Scholar). NO and nitrous acid also induce deamination of Xan and Oxa. the cellular repair mechanism of Xan and Oxa has been H. K. Y. T. Yamada M. Makino K. K. Ide H. Nucleic Acids Res. Scholar). Although Xan and Oxa can genotoxic by directly affecting DNA replication (19Suzuki T. Yoshida M. Yamada M. Ide H. Kobayashi M. Kanaori K. Tajima K. Makino K. Biochemistry. 1998; 37: 11592-11598Google Scholar, R. P.A. K. Nucleic Acids Res. 14: Scholar, H. M. M. H. E. or the stability of duplex DNA T. Matsumura Y. Ide H. Kanaori K. Tajima K. Makino K. Biochemistry. 1997; 36: 8013-8019Google Scholar), in this we have a novel genotoxic mechanism of Oxa that involves DPC formation. Oxa in DNA with proteins such as histone, HMG protein, and DNA glycosylases to form DPCs and The of the DPCs as of DNA and was further by the and (Fig. molecules such as and a of amino acids also with Oxa to form (Fig. The formation of DPCs and was also when containing Oxa was incubated with the extract from HeLa (Fig. Although with (Fig. DPC1 and formed with the HeLa cell extract not the isoforms of formation of DPC1 and was and to and duplex Thus, DNA glycosylases, the proteins in DPC1 and to with Oxa in a DPCs induced by a of nitrous and compounds such as and and and Misra J. Biol. Chem. 1996; and J. Biol. Chem. and These and many of or the between DPCs and carcinogenesis has not been it can be readily from that DPCs formed by NO this and a to of a DNA replication or the of DNA and to T.A. K. Annu. Rev. 2000; Scholar). with with a ability replication Carcinogenesis. 1994; 15: Scholar). with NO and acid is an of and inflammatory responses Biol. 2000; Scholar). formed by the of DPCs between DNA-binding and a P.A. Biochemistry. 2001; Scholar). DPCs also formed in E. to P.A. Biochemistry. 2001; Scholar). DPCs formed by NO and the mechanism of genotoxic and cytotoxic in inflammatory The of has that a of DNA glycosylases Fpg, AlkA, and Endo reacts with Oxa than histone and HMG protein. The of and potentially in DPC formation the the to for the in for the DNA glycosylases and were for DPC formation with the DNA glycosylases, whereas the rate of DPC formation with histone was of (Fig. These results that DPC formation with histone and HMG via DNA-protein whereas DPC formation with the DNA glycosylases via hOGG1, Fpg, AlkA, and Endo a mechanism to the base 2000; Scholar, R. E. J. Biol. Chem. Scholar, T. Y. T. J. 2000; 19: Scholar, R. J. Scholar). Although the of the for Oxa is not Oxa be in the in a similar to a with a amino acid such as or hOGG1, Fpg, and as damage 2000; Scholar, R. E. J. Biol. Chem. Scholar, T. Y. T. J. 2000; 19: Scholar), whereas and Endo III K. H. H. T. H. Y. S. Ide H. Biochemistry. 2000; Scholar, S. T. R. T. S. S. J. Biol. Chem. 1998; 273: Scholar). it has been that Endo not also K. Yamada T. Y. H. Y. S. Ide H. J. Biol. Chem. 2000; Scholar, T. R. M. S. J. Biol. Chem. 2000; Scholar). with an of that and R. S. Carcinogenesis. 1996; 17: Scholar), DPC formation for DNA glycosylases the substrate the of the not have or in the or amino acids be for cross-link formation with Oxa, be the of is for the aspect of DPC this not with the of a of and HMG major in and associated with DNA to structure or to A high of the proteins and the with DNA the reactivity to Oxa in a of damage of DNA glycosylases and a high reactivity to Oxa for the of the in leading to a of Thus, of the of the of DPCs of individual of DPCs and in to NO or nitrous acid. Analysis of the reaction with amino acids and suggested the formation of a between the side of or in and in Oxa (Fig. it has been shown that a is formed between in DNA and amino acid in DNA glycosylases M. J. Am. Chem. Soc. 2001; Scholar, M. Biochemistry. and DNA E. D. B. J. Biol. Chem. Scholar, B. DNA Scholar). is a of abasic lesion formed by oxidation of a abasic Although Oxa and base and have a structure in that is to with nucleophilic which is to form DPC and is used as a also induces cross-links between DNA and proteins in repair B. S. Y. N. Biochemistry. Scholar). Thus, of repair by has a dual DPC formation in and suicide of DNA repair in could pose a threat to the genetic and structural integrity of DNA, hence a carcinogenic We R. S. of and S. of for of protein, an E. for of hNTH1, and

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