Enzymatic Processing of Uracil Glycol, a Major Oxidative Product of DNA Cytosine

Abstract

A major stable oxidation product of DNA cytosine is uracil glycol (Ug). Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo−). During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined. Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in vitro by the E. coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase. The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate. A major stable oxidation product of DNA cytosine is uracil glycol (Ug). Because of the potential of Ug to be a strong premutagenic lesion, it is important to assess whether it is a blocking lesion to DNA polymerase as is its structural counterpart, thymine glycol (Tg), and to evaluate its pairing properties. Here, a series of oligonucleotides containing Ug or Tg were prepared and used as templates for a model enzyme, Escherichia coli DNA polymerase I Klenow fragment (exo−). During translesion DNA synthesis, Ug was bypassed more efficiently than Tg in all sequence contexts examined. Furthermore, only dAMP was incorporated opposite template Ug and Tg and the kinetic parameters of incorporation showed that dAMP was inserted opposite Ug more efficiently than opposite Tg. Ug opposite G and A was also recognized and removed in vitro by the E. coli DNA repair glycosylases, endonuclease III (endo III), endonuclease VIII (endo VIII), and formamidopyrimidine DNA glycosylase. The steady state kinetic parameters indicated that Ug was a better substrate for endo III and formamidopyrimidine DNA glycosylase than Tg; for endonuclease VIII, however, Tg was a better substrate. Free radical-induced DNA damage is a substantial contributor to the spontaneous mutational burden and has been implicated in a variety of disease processes including cancer. Accumulation of free radical-induced DNA damage has also been associated with aging (1Ames B.N. Free Radical Res. Commun. 1989; 7: 121-128Crossref PubMed Scopus (627) Google Scholar, 2Sun Y. Free Radical Biol. Med. 1990; 8: 583-599Crossref PubMed Scopus (1050) Google Scholar, 3Ames B.N. Shigenaga M.K. Hagen T.M. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 7915-7922Crossref PubMed Scopus (5381) Google Scholar). A significant number of free radical-induced mutations produced by oxidizing agents and ionizing radiation are C → T transitions (4Tkeshelashvili L.K. McBride T. Spence K. Loeb L.A. J. Biol. Chem. 1991; 266: 6401-6406Abstract Full Text PDF PubMed Google Scholar, 5McBride T.J. Preston B.D. Loeb L.A. Biochemistry. 1991; 30: 207-213Crossref PubMed Scopus (226) Google Scholar, 6Ayaki H. Higo K. Yamamoto O. Nucleic Acids Res. 1986; 14: 5013-5018Crossref PubMed Scopus (41) Google Scholar, 7Jaberaboansari A. Dunn W.C. Preston R.J. Mitra S. Waters L.C. Radiat. Res. 1991; 127: 202-210Crossref PubMed Scopus (26) Google Scholar, 8Waters L.C. Sikpi M.O. Preston R.J. Mitra S. Jaberaboansari A. Radiat. Res. 1991; 127: 190-201Crossref PubMed Scopus (36) Google Scholar), thus oxidized cytosine residues appear to play an important role in oxidative mutagenesis. Hydroxyl radicals, the principal damaging species produced by oxidizing agents, interact with cytosine residues principally by addition to the 5,6-double bond (9Kuwabara M. Radiat. Phys. Chem. 1991; 37: 691-704Google Scholar, 10Breen A.P. Murphy J.A. Free Radical Biol. Med. 1995; 18: 1033-1077Crossref PubMed Scopus (916) Google Scholar). A major oxidation product of DNA cytosine is cytosine glycol which is unstable and deaminates to uracil glycol (Ug) 1The abbreviations used are: Ug, 5,6-dihydroxy-5,6-dihydrouracil; dUG, 5,6-dihydroxy-5,6-dihydrodeoxyuridine; Tg, 5,6-dihydroxy-5,6-dihydrothymine; dTg, 5,6-dihydroxy-5,6-dihydrodeoxythymidine; dUgTP, 5,6-dihydroxy-5,6-dihydrodeoxyuridine 5′-triphosphate; dTgTP, 5,6-dihydroxy-5,6-dihydrodeoxythymidine 5′-triphosphate; Kf(exo−), DNA polymerase I Klenow fragment lacking proofreading activity; endo, endonuclease; eq, pseudoequatorial; ax, pseudoaxial. 1The abbreviations used are: Ug, 5,6-dihydroxy-5,6-dihydrouracil; dUG, 5,6-dihydroxy-5,6-dihydrodeoxyuridine; Tg, 5,6-dihydroxy-5,6-dihydrothymine; dTg, 5,6-dihydroxy-5,6-dihydrodeoxythymidine; dUgTP, 5,6-dihydroxy-5,6-dihydrodeoxyuridine 5′-triphosphate; dTgTP, 5,6-dihydroxy-5,6-dihydrodeoxythymidine 5′-triphosphate; Kf(exo−), DNA polymerase I Klenow fragment lacking proofreading activity; endo, endonuclease; eq, pseudoequatorial; ax, pseudoaxial. (11Wagner J.R. Hu C.-C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3380-3384Crossref PubMed Scopus (238) Google Scholar). Cytosine glycol can also dehydrate to form 5-hydroxycytosine (5-OHC) (11Wagner J.R. Hu C.-C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3380-3384Crossref PubMed Scopus (238) Google Scholar, 12Dizdaroglu M. Simic M.G. Radiat. Res. 1984; 100: 41-46Crossref PubMed Scopus (37) Google Scholar, 13Teoule R. Int. J. Radiat. Biol. 1987; 51: 573-589Crossref Scopus (364) Google Scholar), while 5-hydroxyuracil (5-OHU) arises from sequential deamination and dehydration (11Wagner J.R. Hu C.-C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3380-3384Crossref PubMed Scopus (238) Google Scholar). Depending on the oxidizing agent used, uracil glycol and 5-hydroxycytosine are formed at comparable levels in DNA and furthermore, the background level of these lesions is high in DNA extracted from untreated cells (11Wagner J.R. Hu C.-C. Ames B.N. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 3380-3384Crossref PubMed Scopus (238) Google Scholar). Oxidized pyrimidine lesions are repaired by a highly conserved process called base excision repair (for reviews, see Refs. 14Demple B. Harrison L. PubMed Scopus Google and S. and the of Scholar). The are recognized by a of called DNA The principal in Escherichia oxidized are endonuclease III (endo and endonuclease VIII (endo Refs. 14Demple B. Harrison L. PubMed Scopus Google and S. and the of Scholar, and formamidopyrimidine DNA glycosylase has also been to oxidized in vitro J. Biol. Chem. Full Text PDF PubMed Google Scholar). of an oxidized pyrimidine by a DNA the bond is the free is by of the by an associated DNA which a in the 14Demple B. Harrison L. PubMed Scopus Google and S. and the of and The an or a be removed by the or of of the 14Demple B. Harrison L. PubMed Scopus Google and S. and the of and in a base which is in by DNA polymerase and by DNA 14Demple B. Harrison L. PubMed Scopus Google and S. and the of and the lesion is repaired to its with the it can DNA polymerase and thus be or it can be bypassed by DNA polymerase and be on its to the pyrimidine lesions the pyrimidine that been to only thymine glycol is a strong blocking lesion to DNA in vitro H. Nucleic Acids Res. PubMed Scopus Google Scholar, Res. Google Scholar, Nucleic Acids Res. 1986; 14: PubMed Scopus Google Scholar, Biochemistry. 1987; PubMed Scopus Google and is in Radiat. Res. PubMed Scopus Google Scholar, E. Res. Google Scholar, J. PubMed Google Scholar). free radical-induced pyrimidine as H. R.J. Biochemistry. 1987; PubMed Scopus (41) Google Scholar), Nucleic Acids Res. PubMed Scopus Google Scholar), and Nucleic Acids Res. PubMed Scopus Google Scholar), DNA and are with A and is a lesion J. J. R. H. Res. 1993; PubMed Scopus Google Scholar). can with A in vitro Nucleic Acids Res. PubMed Scopus Google and has been to be in E. coli L.C. Loeb L.A. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). with vitro Nucleic Acids Res. PubMed Scopus Google and it is from it is a important premutagenic Oxidized from the can also be incorporated these or and are for E. polymerase I Klenow fragment Nucleic Acids Res. PubMed Scopus Google is incorporated in of A and incorporated from the be can be as T and thus be and can also be incorporated is as T to T → G H. S. Loeb L.A. J. Biol. Chem. 1992; Full Text PDF PubMed Google Scholar, H. M. 1992; PubMed Scopus Google Scholar). the of and it to be a substrate for DNA polymerase I Biochemistry. 37: PubMed Scopus Google Scholar). is in to its structural dTgTP, which is a substrate H. R.J. Biochemistry. 1987; PubMed Scopus (41) Google Scholar, Biochemistry. 37: PubMed Scopus Google Scholar). and are incorporated in of T Biochemistry. 37: PubMed Scopus Google and thus be incorporated from oxidized the of Ug in template DNA and that Ug is bypassed by the model enzyme, E. coli DNA polymerase I Kf(exo−), in to Tg which is bypassed in sequence dAMP can be incorporated DNA opposite Ug in DNA is a substrate for III and VIII and to a for it be removed by uracil DNA glycosylase. and were prepared as H. R.J. Biochemistry. 1987; PubMed Scopus (41) Google Scholar, Biochemistry. 37: PubMed Scopus Google the used in the DNA polymerase and the were from was from Klenow fragment DNA and were from U. S. and were from uracil DNA glycosylase was from E. coli endo III endo VIII and DNA glycosylase were as J. Biol. Chem. Full Text PDF PubMed Google Scholar). was from was from oligonucleotides were from or by the on a DNA of used to and used to the oligonucleotides were from oligonucleotides were in the of the by The oligonucleotides were by on a and by in a oligonucleotides were by on an as an The oligonucleotides were with oligonucleotides were a Nucleic Acids the oligonucleotides were with the The oligonucleotides to be used in the were with the and the was removed from of the oligonucleotides with of in a containing and oligonucleotides were a and with oligonucleotides were a and with the to the and oligonucleotides containing a Ug or Tg and were prepared by a of a Nucleic Acids Res. 1993; PubMed Scopus Google of or were for at in of containing dUgTP, or and of The oligonucleotides from the with a or were high on a a of to containing The or Ug or were and DNA or the oligonucleotides or and were by the of or Ug or as a oligonucleotides used for a for of the the of the was the level of by the used for the of were for at in and of DNA the containing the lesion were from the by on or from the and The of oligonucleotides containing or was than containing The of of oligonucleotides lesions was the the of oligonucleotides and of templates and were to a in and with an of uracil DNA glycosylase the A containing an which was used as a substrate for DNA repair was prepared by template with in to in by of the with uracil DNA glycosylase the The of Ug and Tg in sequence contexts was of the a containing the was to a template with Ug or Tg. The was at for with in of the containing and The were used for the the of was and the of of was of the with only was to the of only of the in and The were for at were DNA polymerase the S. the kinetic parameters for lesion a steady state kinetic M. J. J. Biol. Chem. 1987; Full Text PDF PubMed Google was The was prepared by of a containing to and the to of containing and The of Kf(exo−), and of were at and endo endo VIII, and DNA glycosylase the and all the containing the lesion was was at for The were by of and were in a kinetic a template containing Ug or Tg at was to in a for the DNA glycosylase a was to the all the used was endo VIII, and DNA glycosylase were used in the of the were at and with substrate and enzyme, the were to the and the DNA polymerase were by the addition of an of and were by on containing The were in for at and to The in the to the of were a Tg is to be the A by and J. K. DNA on DNA and The of the in which the at the and are in the and and J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google and for a of the of the of Tg formed in and and K. J. Int. J. Radiat. Biol. 1993; PubMed Scopus Google for of the of the and of The of Ug were to be the stable of Ug S. S. and J. and the species was as the from the Biochemistry. 37: PubMed Scopus Google Scholar). The for and parameters for Tg and Ug been Biochemistry. 37: PubMed Scopus Google Scholar). were were on the the of and J. Phys. Chem. 1993; Scopus Google Scholar), and the parameters were from the T. J. Chem. 1995; Scopus Google Scholar). were DNA by the pyrimidine of the base in as Biochemistry. 37: PubMed Scopus Google Scholar), and J. T. and A. of was used to and and of the and were by and of the Nucleic parameters R. S. O. E. R. H. Nucleic Acids Res. 1989; PubMed Scopus Google were the of from is on the of and that the of the used the of the of the DNA the of the and the of for the it to on The of the was to the substantial that from of the in and in a of the of the the A of Tg and Ug on DNA During the the of the glycol has been to be a strong to DNA H. Nucleic Acids Res. PubMed Scopus Google Scholar, Res. Google Scholar, Nucleic Acids Res. 1986; 14: PubMed Scopus Google Scholar, Biochemistry. 1987; PubMed Scopus Google Scholar). Tg was it an to in all sequence Nucleic Acids Res. 1986; 14: PubMed Scopus Google Scholar, J. J. R. H. Res. 1993; PubMed Scopus Google Scholar). the of Ug and Tg by Kf(exo−), DNA templates containing Ug or Tg in the sequence contexts Tg see Refs. Nucleic Acids Res. 1986; 14: PubMed Scopus Google Scholar, H. R.J. Biochemistry. 1987; PubMed Scopus (41) Google Scholar, and J. J. R. H. 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PubMed Scopus Google was and containing Ug or Tg were to or which to the the were at a that in the sequence contexts used for and was Ug or only dAMP was incorporated opposite Ug or Tg all The incorporation of dAMP opposite Ug and Tg in all sequence contexts was to be opposite T in the for was with templates and significant of were opposite T of and incorporation opposite T were also and to of T with or incorporated opposite the levels of for sequence were Nucleic Acids Res. PubMed Scopus Google Scholar). is in to the with all only A was in the product opposite T in of opposite T was also at of template to a opposite Ug and Tg at or of template or was the of Ug or Tg in the template or more was to the level of incorporation opposite Ug and Tg. was with T in the template only opposite Ug or Tg in the template for all opposite T was in the sequence of template of incorporation opposite Ug from a containing T Tg or Ug in was to in the were and all The were at for The in a of was with the of containing or C at The of incorporation opposite Ug was also in a by the that was used to the base pairing of S. M. A.P. 1991; PubMed Scopus Google and the Nucleic Acids Res. PubMed Scopus Google Scholar). with was used to the of S. Y. A.P. Biochemistry. 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Chem. 1987; Full Text PDF PubMed Google state kinetic parameters for of dAMP opposite template Tg, or Ug in the sequence by for incorporation of was to and the are to opposite opposite opposite for incorporation of was to and the are to in a The from I that dAMP was inserted more efficiently opposite Ug than opposite Tg with the of of dAMP opposite Ug in sequence only than opposite T. The of dAMP opposite Tg was than opposite T. The of of dAMP opposite Ug with T was to the the for of dAMP opposite T and Ug were the of the the of dAMP incorporation was to the the for of dAMP opposite T and Tg were the are in with in A with the template the of dAMP opposite Ug and Tg was than opposite T. The of the was by the of with or opposite template with the Tg sequence with Tg, Ug, or T in was to template was by and that opposite Ug was by a of was also to of the or of opposite Tg was of and opposite T was the to the blocking were to template containing Ug, or Tg at and in the of and The of all to the template was was from and and template containing Ug at with in the showed substantial and of T in the in addition to of significant of and was these that only A a stable with Ug and that the was more than an than a The of used for opposite template the sequence of the and of opposite Tg. at of in a was of thymine glycol in the a containing with Tg in the was to be of polymerase were used a of polymerase was used in the in was than of opposite was as the polymerase was of opposite Tg in while at the of opposite Tg in was at polymerase used that the polymerase in a has a better of the thymine glycol lesion in the sequence the is to a at the lesion III and VIII been to a of oxidized in to in to repair these lesions (for a see S. and the of Scholar). also can oxidized J. Biol. Chem. Full Text PDF PubMed Google Scholar). the of endo endo VIII, and on containing Ug opposite A and Ug opposite The that Ug opposite the substrate from oxidation of cytosine in and Ug opposite the substrate from incorporation of from the were by all the from (endo or from (endo VIII and were the for on all state kinetic parameters for the with and that Ug was a better substrate for endo III and than Tg, while Tg was a better substrate for endo endo III and the Ug substrate was and better than the Tg for endo VIII, the Tg substrate was better than the Ug substrate. by of the product endo III of the Ug substrate showed it to a to uracil glycol state kinetic parameters for and by E. coli DNA for of substrate was to and the are to substrate for of substrate was to and the are to in a DNA glycosylase Ug from or with by a at is in to DNA which is a substrate for uracil DNA glycosylase J. Biol. Chem. Full Text PDF PubMed Google Scholar). J. Biochemistry. 1987; PubMed Scopus (41) Google and K. J. R. R. 1995; PubMed Scopus Google the of the of Tg in DNA by a model of DNA containing Tg and J. Biochemistry. 1987; PubMed Scopus (41) Google or K. J. R. R. 1995; PubMed Scopus Google with of DNA containing only a of the of the of Tg and Ug on with Ug and Tg in the Tg sequence can be with the in the the in Tg a significant in the the Tg and the with to while Ug a major oxidative product of DNA in template DNA to be bypassed by and Furthermore, only dAMP was inserted opposite Ug by in sequence contexts and in and and with the sequence showed that the of dAMP opposite Ug was only than opposite T in to of dAMP opposite Tg which was than opposite T with Ug were also by and that the is The major the of a lesion are its to be bypassed by DNA and the of the lesion to The lesion that is uracil from DNA cytosine which is to DNA and with A. uracil which is also from DNA is bypassed at coli DNA polymerase and at the with that it be a premutagenic it that uracil glycol be removed from oxidized DNA to The in and that Ug is recognized and removed in vitro by the E. coli DNA that on oxidized DNA is a substrate for endonuclease better than its lesion thymine however, Ug is efficiently removed than thymine glycol by endonuclease also Ug to be a substrate for endo III J. 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S. and by polymerase J. 1990; PubMed Scopus Google is by → it be that the sequence of Tg is in the of The the Tg and was at polymerase the polymerase was the from the lesion of polymerase were to of thymine glycol the was opposite base or base the lesion at high polymerase from a was at the at the of with uracil glycol and thymine glycol that in all sequence uracil glycol was more bypassed by than thymine that the of the Tg the the and the Furthermore, in sequence thymine glycol was more bypassed than in sequence that the DNA sequence the of the lesion to the DNA be polymerase it has been that Tg is in the for it is a strong for polymerase J. J. R. H. Res. 1993; PubMed Scopus Google Scholar). sequence on lesion been with lesions than Tg S. A.P. J. Biol. Chem. 1993; Full Text PDF PubMed Google and M. H. L. R. DNA on DNA and The of Scholar, for a see S. DNA and DNA in and and also and of a base a and mutational with a number of lesions sequence on lesion and repair for the strong sequence (for a see S. DNA and DNA in and Scholar). The of DNA from templates containing Tg or Ug can be in the of a structural model on the of J. Biochemistry. 1987; PubMed Scopus (41) Google and K. J. R. R. 1995; PubMed Scopus Google Scholar). the A of Tg or Ug is or the at is the of the J. Biochemistry. 1987; PubMed Scopus (41) Google on that a bond and of a in the and that sequence of polymerase of and DNA in the of and are with the of a of the with the at of a thymine to the lesion in or in a significant of the base to the Tg and Ug in the to that in the to that on the of the template in a of opposite the lesion it with of Tg the formed is with the in and that the of product that is base of the lesion is in the for Tg and Ug than in the contexts containing a to the lesion and The significant structural the A of Tg and Ug is the in Tg, which be the of the template in a base and J. Biochemistry. 1987; PubMed Scopus (41) Google and K. J. R. R. 1995; PubMed Scopus Google that in Tg with the of a base to the J. Biochemistry. 1987; PubMed Scopus (41) Google a in the of the base to the lesion, and K. J. R. R. 1995; PubMed Scopus Google a in the associated with the base with that of as as a more of the base to Tg and unstable in the to Tg. be that the at in Ug with the base on the to the that a is to that the of to the lesion in the template in a of of the base opposite Tg to Ug, as by J. Biochemistry. 1987; PubMed Scopus (41) Google Scholar). is with the in and Ug is in bypassed more efficiently than Tg. of the high of DNA which is to DNA polymerase that the and the base in the template are the and the of the polymerase S. S. T. PubMed Scopus Google Scholar). is to that the of opposite Tg is only in the sequence that is to the of Tg. is that in the of the base opposite the to the significant the of to Tg. the that uracil glycol is to be a premutagenic lesion and that thymine it is bypassed by uracil glycol the free radical-induced DNA base lesions as and as with the of the and of base and of the oxidative base lesions are and glycol to be a in The was in the are to the in the and to for the and