Fidelity of Nucleotide Insertion at 8-Oxo-7,8-dihydroguanine by Mammalian DNA Polymerase δ

Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase δ (pol δ) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol δ preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K m). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol δ preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol δ replication fidelity at 8-oxoG depends upon contributions from K m, KddNTP, and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts. Nucleotide insertion opposite 8-oxo-7,8-dihydroguanine (8-oxoG) by fetal calf thymus DNA polymerase δ (pol δ) was examined by steady-state and pre-steady-state rapid quench kinetic analyses. In steady-state reactions with the accessory protein proliferating cell nuclear antigen (PCNA), pol δ preferred to incorporate dCTP opposite 8-oxoG with an efficiency of incorporation an order of magnitude lower than incorporation into unmodified DNA (mainly due to an increased K m). Pre-steady-state kinetic analysis of incorporation opposite 8-oxoG showed biphasic kinetics for incorporation of either dCTP or dATP, with rates similar to dCTP incorporation opposite G, large phosphorothioate effects (>100), and oligonucleotide dissociation apparently rate-limiting in the steady-state. Although pol δ preferred to incorporate dCTP (14% misincorporation of dATP) the extension past the A:8-oxoG mispair predominated. The presence of PCNA was found to be a more essential factor for nucleotide incorporation opposite 8-oxoG adducts than unmodified DNA, increased pre-steady-state rates of nucleotide incorporation by >2 orders of magnitude, and was essential for nucleotide extension beyond 8-oxoG. pol δ replication fidelity at 8-oxoG depends upon contributions from K m, KddNTP, and rates of phosphodiester bond formation, and PCNA is an important accessory protein for incorporation and extension at 8-oxoG adducts. High fidelity DNA replication is critical to the preservation of genomic stability and the avoidance of mutations that can disrupt the regulation of complex biological systems. Cells contain several DNA polymerases and complex DNA repair systems to preserve genomic integrity (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman, New York1992Google Scholar, 2Friedberg E. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society of Microbiology, Washington, D. C.1995Google Scholar). Accurate replication is disrupted by the presence of covalent DNA-chemical adducts, which can be misread and lead to mutations and cancer (3Searle C.E. Chemical Carcinogens. 1 and 2. American Chemical Society, Washington, D. C.1984Google Scholar). Understanding the miscoding events induced by modified DNA is important in understanding risks of environmental chemicals, as well as aspects of chemotherapeutic treatment. Misincorporation is primarily a kinetic phenomenon and not simply thermodynamic. Work with several DNA adducts and artificial DNA bases clearly indicates that both the identity of incorporated bases and their frequency of substitution are functions of which polymerase is used as a catalyst (4Morales J.C. Kool E.T. J. Am. Chem. Soc. 2000; 122: 1001-1007Crossref PubMed Scopus (106) Google Scholar, 5Thomas D.C. Roberts J.D. Sabatino R.D. Myers T.W. Tan C.K. Downey K.M. So A.G. Bambara R.A. Kunkel T.A. Biochemistry. 1991; 30: 11751-11759Crossref PubMed Scopus (94) Google Scholar, 6Johnson R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (696) Google Scholar, 7Langouët S. Müller M. Guengerich F.P. Biochemistry. 1997; 36: 6069-6079Crossref PubMed Scopus (66) Google Scholar, 8Langouët S. Mican A.N. Müller M. Fink S.P. Marnett L.J. Muhle S.A. Guengerich F.P. Biochemistry. 1998; 37: 5184-5193Crossref PubMed Scopus (65) Google Scholar, 9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar, 10Kim M-S. Guengerich F.P. Chem. Res. Toxicol. 1998; 11: 311-316Crossref PubMed Scopus (24) Google Scholar). Our own work on how polymerases influence misincorporation has been focused on 8-oxoG 1The abreviations used are: 8-oxoG, 8-oxo-7,8-dihydrodeoxyguanosine; pol, polymerase; PCNA, human proliferating cell nuclear antigen; G, guanine; A, adenosine; dNTP, deoxynucleotide triphosphate; αS-dNTP, α-thio-substituted dNTPs; bis-Tris-HCl, bis(2-hydroxyethyl)iminotris(hydroxymethyl)methane-HCl; HIV-1 RT, human immunodeficiency virus-1 reverse transcriptase. (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar, 11Lowe L.G. Guengerich F.P. Biochemistry. 1996; 35: 9840-9849Crossref PubMed Scopus (89) Google Scholar, 12Furge L.L. Guengerich F.P. Biochemistry. 1997; 36: 6475-6487Crossref PubMed Scopus (98) Google Scholar, 13Furge L.L. Guengerich F.P. Biochemistry. 1998; 37: 3567-3574Crossref PubMed Scopus (33) Google Scholar, 14Furge L.L. Guengerich F.P. Biochemistry. 1999; 38: 4818-4825Crossref PubMed Scopus (42) Google Scholar). 8-OxoG is a relatively simple adduct in that the only chemical attached to the DNA is one atom of oxygen, and it was selected as a model because of its relatively high mutagenicity and lack of polymerase blockage. This lesion is generally regarded as being the most abundant of those induced by oxidative damage (15Kuchino Y. Mori F. Kasai H. Inoue H. Iwai S. Miura K. Ohtsuka E. Nishimura S. Nature. 1987; 327: 77-79Crossref PubMed Scopus (734) Google Scholar, 16Ames B.N. Gold L.S. Mutation Res. 1991; 250: 3-16Crossref PubMed Scopus (686) Google Scholar, 17Kasai H. Crain P.F. Kuchino Y. Nishimura S. Ootsuyama A. Tanooka H. Carcinogenesis. 1986; 7: 1849-1851Crossref PubMed Scopus (917) Google Scholar). Polymerases derived from prokaryotic systems have been used extensively as models for mechanistic studies because of their availability, the general lack of need for complex accessory proteins, and the availability of structural and mechanistic information (18Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (509) Google Scholar). The question arises as to how relevant findings made with these enzymes are to mammalian and other eukaryotic those polymerases that past of DNA studies the presence of polymerases in both and that are in DNA R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (696) Google Scholar, Nature. 1998; PubMed Scopus Google Scholar, M. J. M. S. A. 1998; PubMed Scopus Google Scholar). the to which the mammalian are to past chemical other than is and the question of how with DNA polymerases is used calf thymus DNA pol to be the DNA polymerase (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman, New York1992Google Scholar, 2Friedberg E. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society of Microbiology, Washington, D. C.1995Google in a of steady-state and pre-steady-state kinetic and that the of the similar to those in the prokaryotic models H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). the of adducts work has been on the replication of mammalian pol δ past DNA adducts and Res. PubMed Scopus Google that the presence of the accessory protein PCNA pol δ of as by similar S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google that PCNA pol δ incorporation and at and 8-oxoG and not The in the of was for 8-oxoG more for The was to extension of the from by a pol by other not be work by M. Tan C.K. Downey K.M. J. Chem. 1996; PubMed Scopus Google that the presence of PCNA to more in the incorporation of in unmodified a in of a DNA for pol δ due to PCNA M. Tan C.K. Downey K.M. J. Chem. 1996; PubMed Scopus Google Scholar). the incorporation of dCTP and opposite 8-oxoG in an oligonucleotide complex with calf thymus pol δ and human PCNA, steady-state and pre-steady-state kinetic The a on the presence of PCNA for the and extension beyond the and the effects of phosphorothioate substitution of that the rate-limiting be phosphodiester bond and from and as (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar, H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google or from from The from S. and and from was and as (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar). fetal calf thymus was from and the of used for the of pol δ are H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). The human was a of from E. PCNA was in and to as K. PubMed Scopus Google with H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). The of PCNA was by the of pol δ was from fetal calf thymus as by A. Res. PubMed Scopus Google with H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). The of pol δ was by analysis of the of pol δ in the by and to a H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). The the and H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). The protein is the of pol δ M. Res. 2000; PubMed Google the protein is an accessory protein to be to PCNA M. H. A. Tan C.K. Downey K.M. Biochemistry. 1996; 35: PubMed Scopus Google and the protein is a of the of the of and has been found in thymus pol δ H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar, M. Res. 2000; PubMed Google Scholar, J. J. Chem. 1998; PubMed Scopus Google Scholar). In a M. Res. 2000; PubMed Google found that the polymerase to the as the for replication factor incorporation of into DNA, in of the work of J. J. Chem. 1998; PubMed Scopus Google Scholar). The of pol δ in the reactions in by the of or with and and to the in a of (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google 8-oxoG. 8-oxoG. in a pol δ of the of pol δ) was to a or of and The reactions with the of an of and and at of or pol PCNA, and 1 in The reactions at in and with of The by and the of was a and by a In steady-state reactions in the of PCNA, the pol δ was increased to and to similar was used for incorporation of opposite and oligonucleotide to the was used with a or in the of the (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar). The of pol δ and PCNA and in the in the presence of of for or and the as for other steady-state Pre-steady-state a by rapid of in with a δ with or PCNA at The of the pol δ on the of pol 1 or PCNA, and The reactions with of at from to The as for steady-state pol of nucleotide was by a of the to the and steady-state of nucleotide The kinetic K for dCTP or to pol was by pre-steady-state The of the pre-steady-state rates was examined by the of and the pre-steady-state rates of incorporation into the The of pol as The pre-steady-state rates and the was to the to K K.A. J. Chem. PubMed Google Scholar). The K for pol δ to or in the presence of PCNA by pre-steady-state rapid quench The DNA was and the in the to and the K for was by the the of DNA and the to the and K dissociation for the The kinetics of of the DNA to as a of dCTP or a large of to pol δ The steady-state for dCTP or incorporation opposite 8-oxoG in the presence of PCNA similar to for dCTP incorporation into unmodified DNA H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). K for dCTP and incorporation opposite 8-oxoG to be and an order of magnitude than the K for nucleotide incorporation into unmodified DNA, the a of can be with of the of these nucleotide incorporation as misincorporation The for incorporation of dCTP opposite 8-oxoG is in a frequency of which to misincorporation kinetic for pol nucleotide nucleotide 8-oxoG. in a PCNA has been to the of pol δ replication and the of and DNA adducts, as 8-oxoG, and Res. PubMed Scopus Google Scholar, S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google Scholar). PCNA was for dCTP and incorporation opposite 8-oxoG was either similar or lower and than in reactions PCNA The K and and for dCTP and studies with other polymerases in the of 8-oxoG with and A, on the 8-oxoG was in the DNA or the nucleotide (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar). studies with pol in the presence of The steady-state for incorporation of opposite and and in the of the prokaryotic polymerases (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google the K for incorporation of than for incorporation of dCTP or opposite 8-oxoG, incorporation opposite and A. the efficiency of incorporation is orders of magnitude lower than for dCTP or incorporation opposite 8-oxoG. misincorporation frequency of can be with the of of nucleotide incorporation to misincorporation opposite This is an order of magnitude lower than for incorporation opposite 8-oxoG, the of the misincorporation frequency is lower than for other polymerases for E. pol (9Einolf H.J. Schnetz-Boutaud N. Guengerich F.P. Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google Scholar). Pre-steady-state kinetic analysis of dCTP or incorporation opposite 8-oxoG by pol δ was to contributions of in the important for fidelity of incorporation opposite 8-oxoG. The of can be by rapid quench kinetics and rates of and phosphodiester bond The incorporation of dCTP or into the by pol δ in the presence of PCNA showed biphasic to an by a The the of nucleotide incorporation in the of the pol was to be for dCTP incorporation and similar to the of dCTP incorporation opposite unmodified The of the to the of in the The was dCTP incorporation and for that and of the pol δ is in an in these upon the in reactions pol δ for dCTP incorporation opposite unmodified G, or from of pol δ not PCNA was from the pre-steady-state of dCTP or incorporation opposite 8-oxoG was at pol δ of The rates of in the of PCNA and for dCTP and orders of magnitude than with PCNA and that PCNA is essential for of an pol δ complex of nucleotide incorporation opposite 8-oxoG. The of was examined for a phosphorothioate to the of a nucleotide incorporation is by phosphodiester bond of the of a with a phosphorothioate can the of pol by the of is a of the The rates of dCTP and incorporation by pol δ in the presence of PCNA by a factor of The are to that phosphodiester bond be the rate-limiting for both dCTP and incorporation opposite 8-oxoG and that the of is a of chemical Pre-steady-state kinetic analysis of the insertion beyond or A:8-oxoG was examined to pol δ has a for extension of either In the presence of PCNA pol δ the A:8-oxoG pol an order of magnitude than pol 1 PCNA was rates of insertion for A:8-oxoG and for and of nucleotide incorporation The pre-steady-state of nucleotide incorporation into the was as a of the of dCTP not or The for DNA modified with 8-oxoG was and the was are than the for dCTP to unmodified DNA H.J. Guengerich F.P. J. 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PCNA was found to be an essential for nucleotide incorporation opposite 8-oxoG and extension beyond the In steady-state reactions with PCNA, the efficiency of nucleotide incorporation by pol δ was by at a factor of for and dCTP incorporation opposite 8-oxoG with reactions with unmodified This in nucleotide insertion efficiency at 8-oxoG is the orders of magnitude of the with prokaryotic polymerases L.G. Guengerich F.P. Biochemistry. 1996; 35: 9840-9849Crossref PubMed Scopus (89) Google Scholar, 12Furge L.L. Guengerich F.P. Biochemistry. 1997; 36: 6475-6487Crossref PubMed Scopus (98) Google Scholar). The in insertion efficiency at 8-oxoG adducts is by an increased K for dCTP or insertion of is because of the of the of polymerases (18Johnson K.A. Annu. Rev. Biochem. 1993; 62: 685-713Crossref PubMed Scopus (509) Google Scholar). In the of PCNA, the efficiency of dCTP incorporation opposite unmodified or 8-oxoG was primarily at the The K of dCTP incorporation opposite unmodified and 8-oxoG increased by a factor of and the was not as The of PCNA the insertion efficiency of pol δ by and for dCTP incorporation opposite and 8-oxoG, that PCNA is an important factor for incorporation of the nucleotide not only opposite unmodified bases more for insertion opposite DNA adducts, as 8-oxoG. The steady-state is by the of dissociation from the DNA or other formation, as clearly by the kinetics in the biphasic pre-steady-state of the 1 and is to that in the or presence of PCNA, the is similar to that for nucleotide incorporation opposite or 8-oxoG This that the of dissociation is not by PCNA in nucleotide incorporation in the of the misincorporation of opposite 8-oxoG, is PCNA is that the is either at a or that other in the have been and that PCNA be more important for the of pol δ in misincorporation than incorporation reactions S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google Scholar). the not a that PCNA primarily by the K for the complex pol δ and DNA L. M. Tan Downey K.M. J. Chem. 1993; PubMed Google Scholar, W. J. L. J. 1991; PubMed Scopus Google Scholar, Tan C.K. Downey K.M. So A.G. Biochemistry. PubMed Scopus Google a be in the presence of The lack of a of nucleotide incorporation PCNA for both dCTP and incorporation reactions opposite 8-oxoG indicates that the rate-limiting is at or to phosphodiester bond The lower for incorporation with dCTP that be in or for the misincorporation of nucleotide incorporation opposite 8-oxoG for incorporation of either dCTP or pol and with rates similar to dCTP incorporation opposite unmodified The in the efficiency of nucleotide insertion opposite 8-oxoG with unmodified is due to an in in with dCTP insertion opposite G, and for dCTP and incorporation opposite 8-oxoG, and to in pol δ incorporated dCTP more than The frequency was to be which to misincorporation of opposite 8-oxoG kinetic for pol δ misincorporation nucleotide misincorporation misincorporation misincorporation H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google nucleotide in a 8-oxoG. PCNA increased the of the pol δ for unmodified DNA by only in work H.J. Guengerich F.P. J. 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Biochemistry. 1996; 35: PubMed Scopus Google that the of PCNA of pol δ is an increased of pol δ for DNA as how these to events in is in steady-state the was in nucleotide incorporation H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google Scholar). a of PCNA on as by S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google Scholar, M. Tan C.K. Downey K.M. J. Chem. 1996; PubMed Scopus Google In in which incorporation is is by in steady-state kinetic in which a is is of PCNA on M. H. A. Tan C.K. Downey K.M. Biochemistry. 1996; 35: PubMed Scopus Google the the pol complex to be in the presence of PCNA, a In that work the not in can be which is orders of magnitude than the in own indicates that the presence of the pol complex L. M. Tan Downey K.M. J. Chem. 1993; PubMed Google Scholar). as in S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google Scholar, that PCNA beyond 8-oxoG The of by PCNA was by S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google to be In pre-steady-state the of incorporation beyond either or by PCNA was The of by PCNA have been in the steady-state work S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google because of the of the of pol δ H.J. Guengerich F.P. J. Chem. 2000; PubMed Scopus Google which can steady-state for the is that S. Tan C.K. Downey K.M. S. A. 1997; PubMed Scopus (97) Google used a of in their the of extension beyond an in the presence of PCNA have been because of the lower efficiency of incorporation of opposite 8-oxoG The of PCNA is more complex than simply the of pol δ with is that PCNA the rates of of the pol complex to and from an analysis be to the is that of other accessory of pol δ have been J. Chem. 1998; PubMed Scopus Google and and Tan C.K. Downey K.M. So A.G. Biochemistry. PubMed Scopus Google have that an and be with mammalian pol as well as the and and PCNA L. J. J. Chem. 2000; PubMed Scopus Google Scholar, J. L. A. N. 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Biochemistry. 1998; 37: 13300-13312Crossref PubMed Scopus (97) Google the kinetic efficiency for incorporation opposite or is the of an that the of the for by is similar to the in DNA, that of the is (15Kuchino Y. Mori F. Kasai H. Inoue H. Iwai S. Miura K. Ohtsuka E. Nishimura S. Nature. 1987; 327: 77-79Crossref PubMed Scopus (734) Google Scholar, 16Ames B.N. Gold L.S. Mutation Res. 1991; 250: 3-16Crossref PubMed Scopus (686) Google Scholar, 17Kasai H. Crain P.F. Kuchino Y. Nishimura S. Ootsuyama A. Tanooka H. Carcinogenesis. 1986; 7: 1849-1851Crossref PubMed Scopus (917) Google Scholar, J. M. J. H. D. M. and Scholar). The has not been are K. a efficiency for incorporation that of by pol δ the of be The of the in mammalian systems K. M. H. M. J. Chem. 1993; PubMed Google is an in that the to to be the that the critical for not be a an is that other mammalian polymerases with high have the replication events at 8-oxoG by mammalian pol δ because is general that is the polymerase in DNA replication (1Kornberg A. Baker T.A. DNA Replication. W. H. Freeman, New York1992Google Scholar, 2Friedberg E. Walker G.C. Siede W. DNA Repair and Mutagenesis. American Society of Microbiology, Washington, D. C.1995Google Scholar). of identity of the of the is the used is a of the in which with pol and other the a of the of pol δ with DNA and is that a of relatively polymerases have been and important contributions to the of the of modified DNA R.E. Prakash S. Prakash L. Science. 1999; 283: 1001-1004Crossref PubMed Scopus (696) Google Scholar, S. A. 2000; PubMed Scopus Google Scholar). it is not which of these in replication past 8-oxoG in mammalian