The N2-Ethylguanine and the O6-Ethyl- and O6-Methylguanine

The effects of N2-ethylGua, O6-ethylGua, and O6-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases α and η have been i...
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Chem. Res. Toxicol. 2003, 16, 1616-1623

The N2-Ethylguanine and the O6-Ethyl- and O6-Methylguanine Lesions in DNA: Contrasting Responses from the “Bypass” DNA Polymerase η and the Replicative DNA Polymerase r Fred W. Perrino,*,† Patrick Blans,‡ Scott Harvey,† Stacy L. Gelhaus,‡ Colleen McGrath,‡ Steven A. Akman,§ G. Scott Jenkins,§ William R. LaCourse,‡ and James C. Fishbein*,‡ Department of Biochemistry and Department of Cancer Biology, Wake Forest University Health Sciences, Winston-Salem, North Carolina 27157, and Department of Chemistry and Biochemistry, University of Maryland, Baltimore County, 1000 Hilltop Circle, Baltimore, Maryland 21250 Received July 31, 2003

The effects of N2-ethylGua, O6-ethylGua, and O6-methylGua adducts in template DNA on polymerization by mammalian DNA polymerases R and η have been investigated. The N2ethylGua adduct blocks polymerization by the replicative DNA polymerase R to a much greater extent than does the O6-ethyl- or the O6-methylGua adducts. The DNA polymerase η efficiently and accurately bypasses the N2-ethylGua lesion but like DNA polymerase R is similarly blocked by the O6-ethyl- or the O6-methylGua adducts. A steady state kinetic analysis of nucleotide insertion opposite the N2-ethylGua and the O6-ethylGua adducts by the DNA polymerases R and η and extension from 3′-termini positioned opposite these adducts was performed to measure the efficiency and the accuracy of DNA synthesis past these lesions. This analysis showed that insertion of Cyt opposite the N2-ethylGua adduct by DNA polymerase R is approximately 104-fold less efficient than insertion of Cyt opposite an unadducted Gua residue at the same position. Extension from the N2-ethylGua:Cyt 3′-terminus by DNA polymerase R is approximately 103-fold less efficient than extension from a Cyt opposite the unadducted Gua. Insertion of Cyt opposite the N2-ethylGua lesion by the DNA polymerase η is about 370fold more efficient than by the DNA polymerase R, and extension from the N2-ethylGua:Cyt 3′-terminus by the DNA polymerase η is about 3-fold more efficient than by the DNA polymerase R. Furthermore, the DNA polymerase η preferably inserts the correct nucleotide Cyt opposite the N2-ethylGua lesion with nearly the same level of accuracy as opposite an unadducted Gua, thus minimizing the mutagentic potential of this lesion. This result contrasts with the relatively high misincorporation efficiency of Thy opposite the O6-ethylGua adduct by the DNA polymerases R and η. In reactions containing both DNA polymerases R and η, synthesis past the N2-ethylGua adduct is detected to permit completed replication of the adducted oligonucleotide template. These results suggest that accurate replication past the N2-ethylGua adduct might be facilitated in cells by pausing of replication catalyzed by DNA polymerase R and lesion bypass catalyzed by DNA polymerase η.

Introduction Covalent modification of DNA can give rise to cell death or mutation; the ultimate outcome is dictated by a complex web of factors among which are sequence and atom site of adduct deposition and interactions with DNA pols1 and DNA repair pathways. Considerable progress has been made regarding understanding the biological effects of simple methylating and ethylating agents of many types, including the diazonium ions that arise from * To whom correspondence should be addressed. (F.W.P) Tel: 336716-4349. Fax: 336-716-7200. E-mail: [email protected]. (J.C.F.) Tel: 410-455-2190. E-mail: [email protected]. † Department of Biochemistry, Wake Forest University Health Sciences. ‡ Department of Chemistry and Biochemistry, University of Maryland. § Department of Cancer Biology, Wake Forest University Health Sciences. 1 Abbreviations: DNA pol, DNA polymerase; MBP, maltose binding protein.

carcinogenic nitrosamines that are widely distributed in the human environment. These types of agents give rise to adducts at endocyclic heteroatoms and the exocyclic oxygen atoms of DNA bases. It is largely these latter lesions that are responsible for the mutagenic activities (1-5) of these agents since they give rise with high frequency to misincorporation by some DNA pols (6-11). However, a new class of DNA pols has recently been identified (reviewed in ref 12) that might serve to alleviate replication blocks and minimize mutagenesis through high fidelity synthesis past unrepaired lesions. Recently, the novel lesion N2-ethylguanine has been identified in the DNA of ethanol-treated mice (13) and in alcoholic and nonalcoholic humans (14, 16). It has been proposed to arise through oxidation of ethanol to acetaldehyde and biological reduction of the aldehyde adduct (13, 15, 16). Both ethanol and acetaldehyde have been recognized as potential human mutagens and carcinogens (17-24). The correlation between ethanol consumption

10.1021/tx034164f CCC: $25.00 © 2003 American Chemical Society Published on Web 11/13/2003

N2-Ethylguanine and O6-Ethylguanine

and cancers of the aerodigestive tract is well-established (17-21). So, it is of interest to understand the biological impact of the N2-ethylGua lesion and its interaction with the DNA replication apparatus. While our own investigation was underway, the Shibutani group reported that the N2-ethylGua lesion induced miscoding by the exonuclease-free large fragment of Escherichia coli DNA pol I (25). DNA pol I preferentially incorporated Gua and Cyt opposite N2-ethylguanine, indicating the possibility of a high frequency of GuafCyt transversions induced by N2ethylguanine. We are interested in the interaction of mammalian DNA pols with this novel ethanol-derived lesion and, in particular, whether there are differences in the responses of replicative DNA pols vs those of the newly characterized “bypass” DNA pols that might have an impact on the biological manifestations of N2-ethylguanine. We report here the results of a translesion synthesis analysis using the mammalian replicative DNA pol R and the bypass DNA pol η with the N2-ethylguanine lesion, which contrast with what has been reported for the E. coli DNA pol I. The adduct N2-ethylguanine is a powerful block to DNA synthesis catalyzed by DNA pol R, more potent than the promutagenic adducts O6-ethylguanine and O6-methylguanine; however, the adduct N2-ethylguanine is efficiently bypassed by DNA pol η, with an accuracy opposite N2-ethylguanine that is comparable with unadducted guanine. Indeed, we show that DNA pol η “rescues” polymerization when DNA pol R fails to copy past the N2-ethylguanine and allows for efficient polymerization of the N2-ethylguanine-adducted polynucleotide. Finally, the efficient, relatively accurate, bypass of N2ethylguanine by DNA pol η is in stark contrast to both the strong block to this polymerase caused by O6ethylguanine and O6-methylguanine and the high frequency of Thy misinsertion at the O6-ethylguanine. Implications for mutation and mutation avoidance due to replication of these guanine adducts are discussed.

Experimental Procedures Adducted Nucleotides. Suitably protected N2-ethyl and O6ethyl phosphoramidites were prepared by eight step and four step syntheses, respectively, generally following published procedures (contained in the Supporting Information). The former synthesis was initiated with 2′-deoxyguanosine while the latter started with commercially available N2-phenoxyacetyl5′-(p-dimethoxytrityl)-2′-deoxyguanosine. Physical data for the final products are listed below. Schematics outlining the syntheses and step by step procedures and physical characterizations are described in the Supporting Information. Oligomer 4, containing O6-methylGua at a single site, was obtained from Midland Certified Reagent Company (Midland, TX) and purified as described below. N2-Ethyl-3′-(N,N-diisopropyl-2-cyanoethyl)-5′-(p-dimethoxytrityl)-2′-deoxyguanosine Phosphoramidite. 1H NMR (CDCl3): δ 1.08-1.20 (m, 15, CH3 iPr and Et), 2.46, 2.61 (2 t, 2, CH2CN), 2.55-2.72 (m, 2, H2′,2′′), 3.34 (m, 4), 3.55-3.81 (br m, 4), 3.76, 3.77 (2 s, 6, CH3 DMTr), 4.25, 4.70 (2 m, 2, H3′,4′), 6.29 (“t”, 1, J ) 6.3 Hz, H1′), 6.61 (br s, 1, NHCH2), 6.77-7.43 (m, 13, Ar DMTr), 7.65, 7.67 (2 s, 1, H8), 11.97 (br s, 1, NH). 31P NMR (CDCl3): δ 149.698 and 150.133. HRMS: calcd, 798.3744; observed, 798.3744. N2-Phenoxyacetyl-O6-ethyl-3′-(N,N-diisopropyl-2-cyanoethyl)-5′-(p-dimethoxytrityl)-2′-deoxyguanosine Phosphoramidite. 1H NMR (CDCl3): δ 1.09-1.19 (m, 12, CH3 iPr),1.52 (t, 3, J ) 6.9 Hz, CH3CH2O), 2.45, 2.62 (2t, 2, CH2CN), 2.602.90 (m, 2, H2′,2′′), 3.38 (m, 2, H5′,5′′), 3.55-3.82 (m, 4, OCH2 and

Chem. Res. Toxicol., Vol. 16, No. 12, 2003 1617 CH iPr), 3.77 (s, 6, CH3 DMTr), 4.29 (m, 1, H4′), 4.66 (q, 2, J ) 6.9 Hz, CH3CH2O), 4.79 (m, 3, H3′ and CH2 PAC), 6.44 (“t”, 1, J ) 6.3 Hz, H1′), 6.75-7.40 (m, 18, Ar DMTr and PAC), 8.01, 8.02 (2 s, 1, H8), 8.64 (br s, 1, NH). 31P NMR (CDCl3): δ 149.906. HRMS: calcd, 954.3931; observed, 954.3928. Template oligonucleotides, as defined below, were synthesized by Midland Certified Reagents, using phosphoramidite methodology with phenoxyacetyl (PAC)-protected bases, as appropriate. Deprotection entailed incubation with aqueous ammonia for 2 h at room temperature. Oligodeoxynucleotides were purified by reverse phase chromatography prior to receipt.

5′-TCGAGACTTCXAAGGGTTCCGGAGCGGCCAAA-3′ where for 1, X ) Gua; for 2, X ) N2-ethylGua; for 3, X ) O6ethylGua; and for 4, X ) O6-methylGua. Oligonucleotide Purification. Further purification was carried out using ion-pairing reverse phase HPLC. A detailed list of the equipment and gradient conditions is included in the Supporting Information. Analysis. Mass spectral analysis of the final purified materials was carried out at the Iowa High-Resolution Mass Spectrometry Facility by Dr. Lynn Teesch using a Bruker Biflex III MALDI-TOF mass spectrometer (linear TOF) using negative ion detection. The matrix consisted of 15 mg/mL hydroxypicolinic acid in 30/70 acetonitrile/water containing 10 mM ammonium citrate. Instrument calibration was based on external calibration of two known oligonucleotides of masses 6115.01 and 9189.05. Mass assignment was based on the mass of maximum intensity. Mass of 3: Anal, 9925.94; calcd, 9925.54. Mass of 2: Anal, 9926.43; calcd, 9927.54. Purine base analysis was performed by hydrolysis in 0.1 M HCl for 30 min at 80 °C, neutralization, separation, and quantification by HPLC on a Waters 2690 instrument run under Millenium software with diode array detection. A Phenomenex Luna 5 mm C-18 column and gradients employing aqueous 0.01 M ammonium acetate and acetonitrile were used to effect separations. The ratios, based on analyses of duplicate injections, of N2-EtGua/Gua (for 2) and O6-EtGua/Gua (for 3) were calculated from the quantifications based on three point standard curves using authentic standards. Ratio of N2-EtGua/Gua for 2: Anal, 0.104; calcd, 0.100. Ratio of O6-EtGua/Gua for 3: Anal, 0.105; calcd, 0.100. The primer oligonucleotides were synthesized at Wake Forest University Health Sciences. Enzymes. The DNA pol R was affinity purified from calf thymus tissue as the four subunit DNA pol-R-primase complex using the SJK132-20 antibody (26, 27). The concentration of the DNA pol R protein (140-180 kDa) was estimated from SDS silver-stained gels, and the specific activity for the enzyme preparation used in these experiments, estimated to be 10 000 units/mg, was determined as previously described (28). The immunoaffinity-purified DNA pol R is devoid of detectable exonuclease or endonuclease activity. The DNA pol R used in these studies is the enzyme described in ref 28 that has been stored at -80 °C with no apparent loss of activity. The recombinant human DNA pol η was prepared in E. coli as the MBP-DNA pol η fusion protein, and the MBP-DNA pol η protein was used in all of the assays containing DNA pol η. The DNA pol η gene was recovered by reverse transcription polymerase chain reaction of human myeloblast mRNA and cloned into the XbaI-SalI site of the pMal-c2 plasmid (New England Biolabs, Beverly, MA) to generate the pMAL-DNA pol η plasmid. The sequence of the DNA pol η gene was confirmed by automated DNA sequencing. For overexpression, the MBPDNA pol η plasmid was electroporated into Stratagene Codon Plus RP cells. Cells were grown in LB medium at 37 °C to A595 ) 0.5, and isopropyl-1-thio-β-D-galactopyranoside was added to a final concentration of 0.5 mM for 10 min. Cell cultures were transferred to shaking water baths at 15 °C for an additional 20 h. Cell extracts were prepared by sonication in 20 mM TrisHCl (pH 7.5), 1 mM EDTA, and 200 mM NaCl containing a complete protease inhibitor cocktail (Roche, catalog no. 1697498). The MBP-DNA pol η fusion protein was affinity-purified using

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Perrino et al. was used. For nucleotide insertion, the 3′-terminus of the oligomer primer (14-mer) was positioned one nucleotide from the target position, and for extension assays, the 3′-terminus of the oligomer primer (15-mer) was positioned opposite the target. The amounts of DNA pols R and η used in the kinetic assays were determined in control reactions such that less than 20% of the properly base-paired primer termini was extended after a 10 min incubation at 37 °C using the correct dNTP at 0.1 mM. The kinetic assay reaction mixtures contained DNA pol R (approximately 0.15 ng/µL for insertion and 0.04 ng/µL for extension) or DNA pol η (2.0 ng/µL for insertion and 0.75 ng/µL for extension) and varied concentrations of dNTPs as indicated in the figure and table legends. Incubations were 10 min at 37 °C unless noted otherwise. All reactions were stopped in 70% ethanol, dried, and resuspended in 95% formamide. Products were separated by electrophoresis through 23% polyacrylamide-7 M urea gels and quantified by Phosphorimager (Molecular Dynamics) analysis. The initial velocity (vo) values (nmol/min/mg) were determined for each nucleotide concentration by quantifying the amount of primer elongated to product in the 10 min incubation in the presence of the indicated amounts of DNA pol. The vo values were plotted as a function of the dNTP concentration to generate hyperbolic plots. The apparent KM and Vmax values and standard errors were determined by nonlinear regression analysis of the vo vs dNTP concentration plots using SigmaPlot 8.02 (SPSS Science, Inc.).

Results and Discussion

Figure 1. Primer extension reactions (10 µL) by DNA pol R (A) and DNA pol η (B) using the oligonucleotide templates 1, 2, 3, and 4 (X ) Gua, N2-ethylGua, O6-ethylGua, and O6-methylGua, respectively). The template primers in A were incubated with increased amounts of DNA pol R at approximately 0.038 (lanes 2, 8, 14, and 20), 0.075 (lanes 3, 9, 15, and 21), 0.15 (lanes 4, 10, 16, and 22), 0.3 (lanes 5, 11, 17, and 23), and 0.6 ng/µL (lanes 6, 12, 18, and 24). The template primers in B were incubated with increased amounts of DNA pol η at approximately 0.47 (lanes 2, 8, 14, and 20), 0.94 (lanes 3, 9, 15, and 21), 1.9 (lanes 4, 10, 16, and 22), 3.8 (lanes 5, 11, 17, and 23), and 7.5 ng/µL (lanes 6, 12, 18, and 24). Control reactions (A and B: lanes 1, 7, 13, and 19) contained no enzyme. The sequence of the DNA template and the positions of the starting 12 nucleotide primer (12-mer) and oligonucleotide products are indicated. an amylose resin (New England Biolabs) as described by the manufacturer. Glycerol was added to the purified MBP-DNA pol η protein to a final concentration of 10% (v/v), and aliquots were stored at -80 °C. The concentration of the MBP-DNA pol η was determined by A280 using the molar extinction coefficient  ) 130 970 M-1 cm-1 (29). The amount of full-length MBP-DNA pol η (122 kDa) was estimated to be 50% of the total protein. The specific activity of the MBP-DNA pol η used in this study is similar to the activities of DNA pol η reported by others using oligonucleotide template primers (40, 42). An SDSPAGE analysis of the recombinant MBP-DNA pol η protein is included in the Supporting Information. DNA Pol Assays. For the primer extension assays in Figures 1 and 4, the oligomer primer (12-mer) was labeled with 32P at the 5′-position and hybridized to the oligomer templates 1-4 at a 1:1 molar ratio (27) so that the 3′-terminus was positioned three nucleotides from the target position. Reaction mixtures contained 20 mM Tris-HCl (pH 7.5), 5 mM MgCl2, 2 mM DTT, 0.1 mg/mL BSA, 100 nM DNA template primer, 0.1 mM dNTPs, and the amounts of DNA pols R and η indicated in the figure legends. Reactions were at 37 °C for 10 min (Figure 1) and for the indicated times in Figure 4. For kinetic assays, the sitespecific insertion and extension procedure of Boosalis et al. (30)

Primer Extension Reactions. Polymerization reactions catalyzed by the replicative DNA pol R using the oligonucleotide templates 1-4 indicate that N2-ethylGua is a strong block to primer extension, stronger than O6ethylGua and O6-methylGua. In four separate reactions, a 5′-32P-labeled 12-mer primer was hybridized to the four DNA templates such that the 3′-terminus was positioned three nucleotides from the target Gua or adducted Gua in the template (Figure 1). Upon addition of increased amounts of the replicative DNA pol R, elongation of the 12-mer primer to generate oligonucleotide products of increased lengths is detected using all four templates. The DNA pol R copies the unadducted template 1 generating products of varied lengths ranging from 13 to 25 nucleotides (Figure 1A, lanes 2-6). Using this specific DNA template, some DNA pol R pausing at unadducted nucleotides is apparent by the accumulation of products 13 and 14 nucleotides in length and to a lesser extent of products 20-22 nucleotides in length. These positions in the DNA template correspond to runs of adjacent purine nucleotides, and pausing by DNA pol R at some purine-rich template sequences has been documented (31, 32). The length of the oligomer products detected using the unadducted template increases with addition of increased amounts of DNA pol R, and at the highest DNA pol R concentration tested, greater than 70% of the 12-mer primer has been elongated past the target Gua site (Figure 1A, lane 6). These reactions demonstrate the ability of DNA pol R to effectively utilize this unadducted oligonucleotide as a template. In contrast, upon addition of the same increased amounts of DNA pol R to reactions containing the N2-ethylGuaadducted template 2, the major oligonucleotide product that accumulates is 14 nucleotides in length corresponding to product generated as a result of blocked polymerization prior to the site of the N2-ethylGua adduct (Figure 1A, lanes 8-12). This product indicates a strong block of the DNA pol R prior to nucleotide insertion opposite the N2-ethylGua adduct in the DNA template. There is no detectable accumulation of a 15 nucleotide product. This

N2-Ethylguanine and O6-Ethylguanine

suggests that DNA pol R elongates nucleotides that might be inefficiently inserted opposite the N2-ethylGua adduct. At the highest DNA pol R concentration tested, less than 1% of the 12-mer primer has been elongated past the target N2-ethylGua adduct (Figure 1A, lane 12). The amount of synthesis by DNA pol R past the N2ethylGua adduct is dramatically lower than synthesis past the O6-ethylGua or the O6-methylGua adducts in these primer extension reactions (Figure 1A). Upon addition of increased amounts of DNA pol R to reactions containing the O6-ethylGua and O6-methylGua-adducted templates 3 and 4, a similar accumulation of products 14 and 15 nucleotides in length is detected demonstrating the similar blocking effects on DNA pol R by these two O6-alkylGua lesions (Figure 1A, compare lanes 14-18 and 20-24). The blocking effect of the O6-methylGua lesion on DNA pols has been described (33-37), and these primer extension reactions demonstrate that the blocking effect of the O6-ethylGua lesion on polymerization by DNA pol R is similar to that of the O6-methylGua lesion. The strong blocking effect of the N2-ethylGua lesion relative to the O6-ethylGua and O6-methylGua lesions is most apparent in reactions containing the largest amounts of DNA pol R used in these primer extension experiments. The DNA pol R elongates approximately 57 and 28% of the 12-mer primer past the O6-ethylGua and O6-methylGua lesions, respectively, as compared to less than 1% of the 12-mer primer past the N2-ethylGua lesion (Figure 1A, compare lanes 18 and 24 to 12). Polymerization catalyzed by the lesion bypass DNA pol η is distinctly different: it copies the N2-ethylGuacontaining template, 2, with an efficiency that is indistinguishable from that with which it copies the normal Gua-containing oligonucleotide, 1, while it is appreciably blocked by the O6-ethylGua in template 3 and the O6methylGua in template 4 (Figure 1B). The addition of increased amounts of DNA pol η in primer extension reactions results in increased elongation of the 12-mer primer past the target Gua in the control template, 1 (Figure 1B, lanes 2-6). Figure 1B, lanes 8-12, shows that the products generated with the same amounts of DNA pol η using the N2-ethylGua-containing template were not appreciably different from those generated using the control Gua-containing template (Figure 1B, lanes 2-6). Surprisingly, polymerization past O6-ethylGua (Figure 1B, lanes 14-18) and O6-methylGua (Figure 1B, lanes 20-24) by DNA pol η was not nearly as efficient as polymerization past N2-ethylGua (Figure 1B, lanes 8-12). Addition of the largest amounts of the DNA pol η used in these primer extension reactions resulted in elongation of about 5% of the primers past the O6ethylGua lesion and 4% of the primers past the O6methylGua lesion as indicated by products greater than 15 nucleotides in length (Figure 1B, lanes 18 and 24). The accumulation of oligomer products at the 14-mer and 15-mer positions indicates reduced polymerization efficiencies by DNA pol η prior to and after nucleotide insertion opposite the O6-ethylGua and O6-methylGua lesions. Steady State Kinetic Assays: The Blocking Potentials of N2-EthylGua and O6-EthylGua. A steady state kinetic assay (30, 38) was used to precisely quantify the blocking effects of the N2-ethylGua and the O6ethylGua adducts by measuring the efficiencies of nucleotide insertion and extension from 3′-termini positioned opposite these adducts by DNA pols R and η in the

Chem. Res. Toxicol., Vol. 16, No. 12, 2003 1619 Table 1. Nucleotide Insertion Opposite N2-EthylGua and O6-EthylGua by DNA Pols r and ηa KM (µM)

Vmax (nmol/ min/mg)

at template Gua pol R dCMP 1.0 ( 0.092 15 ( 0.58 dAMP ND b ND dGMP ND ND dTMP ND ND pol η dCMP 9.8 ( 2.1 1.7 ( 0.18 dAMP 650 ( 190 0.15 ( 0.023 dGMP 140 ( 47 0.072 ( 0.0077 dTMP 1800 ( 640 0.43 ( 0.11 pol R dCMP pol η dCMP dAMP dGMP dTMP

Vmax/KM relative (nmol/min/ insertion mg/M) frequency f 1.5 × 107 1.3 × 102c 6.5 × 102c 5.1 × 102c 1.7 × 105 2.3 × 102c 5.1 × 102c 2.4 × 102c

at template N2-ethylGuae ND ND 8.4 × 102c 3.9 ( 0.40 1.2 ( 0.041 3.1 × 105 520 ( 73 0.098 ( 0.0066 1.9 × 102d 32 ( 27 0.018 ( 0.0030 5.6 × 102d 720 ( 110 0.27 ( 0.023 3.8 × 102d

at template O6-ethylGuae pol R dCMP 970 ( 230 17 ( 1.8 1.8 × 104 dTMP 1100 ( 170 31 ( 2.1 2.8 × 104 pol η dCMP 390 ( 38 0.40 ( 0.017 1.0 × 103 dAMP 610 ( 44 0.35 ( 0.013 5.7 × 102 dTMP 160 ( 25 0.53 ( 0.027 3.3 × 103

1 1/120 000 1/23 000 1/29 000 1 1/740 1/330 1/710

1 1/1600 1/550 1/820 1 1/0.64 1 1/1.8 1/0.30

a The insertion data from Figures 2 and 3 and from the data derived using incorrect dNTPs (not shown) were quantified and analyzed as described in the Experimental Procedures to determine the apparent KM and Vmax values ( standard errors. Frequencies are relative to insertion of dCMP. b ND, not determined. c Rate constant estimated from the slope of the vo vs [dNTP] plot. d Reaction times were 60 min. e For insertion of dAMP, dGMP, and dTMP opposite N2-ethylGua, the rates of insertion by DNA pol R were below the level of detection. For insertion of dAMP and dGMP opposite O6-ethylGua by DNA pol R and for insertion of dGMP opposite O6-ethylGua by DNA pol η, the rates of insertion were below the level of detection. f Relative insertion frequency ) 1/(Vmax/KM correct/Vmax/KM incorrect).

presence of varied concentrations of a single dNTP (Figures 2 and 3). 1. Insertion and Extension Opposite Gua. The efficiencies of polymerization (Vmax/KM) past the unadducted Gua in template 1 by DNA pols R and η was first established by measuring the apparent KM and Vmax values for insertion of dCMP at the target Gua and the apparent KM and Vmax values for extension from the Gua: Cyt 3′-terminus in the presence of increased concentrations of dCTP and dGTP, respectively (Figures 2A,B and 3A,B). Quantification of these data is summarized in Tables 1 and 2. The efficiency of correct nucleotide incorporation is about 88-fold higher for DNA pol R (1.5 × 107 nmol/min/mg/M) as compared to that for DNA pol η (1.7 × 105 nmol/min/mg/M), and the efficiency of extension from the Gua:Cyt 3′-terminus is about 380-fold higher for DNA pol R (2.8 × 108 nmol/min/mg/M) than for DNA pol η (7.4 × 105 nmol/min/mg/M). These data indicate a greater efficiency for polymerization of correct polynucleotide chains past the unadducted Gua by DNA pol R relative to that for DNA pol η. 2. Insertion and Extension Opposite Adducts: DNA Pol r. Consistent with the qualitative results presented in Figure 1A, DNA pol R insertion of Cyt and extension from an adducted Gua:Cyt pair is substantially retarded in the presence of either the N2-ethylGua or the O6-ethylGua lesion. The data from experiments depicted in Figure 2C,D, summarized in Tables 1 and 2, indicate that DNA pol R inserts Cyt opposite N2-ethylGua with a Vmax/KM value (8.4 × 102 nmol/min/mg/M) that is reduced by ∼104 as compared to normal Gua (1.5 × 107 nmol/

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Perrino et al.

Table 2. Extension from 3′-Termini Positioned Opposite N2-EthylGua and O6-EthylGua by DNA Pols r and ηa mispair (template: primer)

KM (µM)

pol R Gua:Cyt 0.43 ( 0.040 Gua:Ade ND b Gua:Thyd 740 ( 110 pol η Gua:Cyt 3.4 ( 0.30 Gua:Ade ND Gua:Thy 240 ( 120 pol R N2eG:Cyt 270 ( 99 N2eG:Thy 730 ( 110 pol η N2eG:Cyt 2.4 ( 0.44 N2eG:Thy ND 6 pol R O eG:Cyt 170 ( 57 O6eG:Ade ND O6eG:Thy 35 ( 12 pol η O6eG:Cyt 9.5 ( 1.1 O6eG:Ade ND O6eG:Gua ND O6eG:Thy 11 ( 3.3

Vmax (nmol/min/ mg) 120 ( 5.0 ND 96 ( 5.7 2.5 ( 0.083 ND 0.11 ( 0.010 77 ( 8.8 8.1 ( 0.45 2.2 ( 0.14 ND 13 ( 1.1 ND 100 ( 14 0.68 ( 0.024 ND ND 0.49 ( 0.037

Vmax/KM relative (nmol/min/ extension mg/M) frequency e 2.8 × 108 6.3 × 102c 1.3 × 105 7.4 × 105 1.1 × 102c 4.6 × 102 2.9 × 105 1.1 × 104 9.2 × 105 3.4 × 102c 7.6 × 104 1.9 × 102c 2.9 × 106 7.2 × 104 5.8 × 101c 1.2 × 102c 4.4 × 104

1 1/440 000 1/2200 1 1/6700 1/1600 1 1/26 1 1/2700 1 1/400 1/0.026 1 1/1200 1/600 1/1.6

a The extension data from Figures 2 and 3 and from the data derived using incorrectly base-paired 3′-termini (not shown) were quantified and analyzed as described in the Experimental Procedures to determine the apparent KM and Vmax values ( standard errors. Frequencies are relative to extension from dCMP. b ND, not determined. c Rate constant estimated from the slope of the vo vs [dGTP] plot. d For extension from Gua:Gua, N2-ethylGua: Ade, and N2-ethylGua:Gua by DNA pols R and η, the rates were below the level of detection. For extension from O6-ethylGua:Gua by DNA pol R, the rate was below the level of detection. e Relative extension frequency ) 1/(Vmax/KM correct/Vmax/KM incorrect).

Figure 2. Kinetic assays for single nucleotide insertion (A, C, and E) and for extension (B, D, and F) polymerase reactions (10 µL) were prepared as described in the Experimental Procedures. The template:primers containing the indicated target nucleotides Gua (G), N2-ethylGua (N2EG), or O6-ethylGua (O6EG) were incubated with DNA pol R in the presence of the indicated concentrations of dCTP or dGTP. The positions of the starting 14 nucleotide primer (14-mer) or 15 nucleotide primer (15-mer) and oligonucleotide products (f) are indicated.

min/mg/M). The efficiency of extension from the N2ethylGua:Cyt pair by DNA pol R (2.9 × 105 nmol/min/

Figure 3. Kinetic assays for single nucleotide insertion (A, C, and E) and for extension (B, D, and F) polymerase reactions (10 µL) were prepared as described in the Experimental Procedures. The template:primers containing the indicated target nucleotides Gua (G), N2-ethylGua (N2EG), or O6-ethylGua (O6EG) were incubated with DNA pol η in the presence of the indicated concentrations of dCTP or dGTP. The positions of the starting 14 nucleotide primer (14-mer) or 15 nucleotide primer (15-mer) and oligonucleotide products (f) are indicated.

mg/M) is reduced by a factor of ∼103 relative to the normal Gua:Cyt pair (2.8 × 108 nmol/min/mg/M). The response of DNA pol R to the ethyl lesion at O6 is quantitatively similar with a Vmax/KM value for insertion of Cyt opposite this lesion (1.8 × 104 nmol/min/mg/M) that is ∼800-fold below what is observed for normal Gua (1.5 × 107 nmol/min/mg/M). The DNA pol R is less active at extension from the O6-ethylGua:Cyt pair (7.6 × 104 nmol/ min/mg/M) by ∼3 × 103 as compared to extension from the normal Gua:Cyt pair (2.8 × 108 nmol/min/mg/M). Thus, in the case of DNA pol R, ethyl adducts at both the N2 and the O6 position present a substantial obstruction to insertion of Cyt with reduced polymerization efficiencies of 103-104, the N2-ethyl lesion being a more effective block by a factor of ∼10. Extension from Cyt paired with the adducts is approximately equally sluggish, being ∼1000-fold slower than with the normal pair. 3. Insertion and Extension Opposite Adducts: DNA Pol η. In contrast to DNA pol R, DNA pol η processes the two ethylated guanine lesions with distinct differences. From experiments depicted in Figure 3C,D and summarized in Tables 1 and 2, DNA pol η inserts Cyt opposite N2-ethyl Gua and extends from the N2ethylGua:Cyt pair slightly more efficiently than insertion opposite Gua and extension from the normal Gua:Cyt pair! The Vmax/KM value for insertion of Cyt opposite N2ethylGua by DNA pol η (3.1 × 105 nmol/min/mg/M) indicates a ∼370-fold greater efficiency than by DNA pol R (8.4 × 102 nmol/min/mg/M), while extension from the N2-ethylGua:Cyt by DNA pol η (9.2 × 105 nmol/min/mg/ M) is ∼3-fold more efficient than for DNA pol R (2.9 ×

N2-Ethylguanine and O6-Ethylguanine

105 nmol/min/mg/M). In contrast, the O6-ethylGua lesion remains an obstruction for DNA pol η (Figure 3 E,F and Tables 1 and 2): insertion efficiency of Cyt (1.0 × 103 nmol/min/mg/M) is 170-fold lower than opposite Gua (1.7 × 105 nmol/min/mg/M), but efficiency of extension from the O6-ethylGua:Cyt pair (7.2 × 104 nmol/min/mg/M) is just ∼10-fold lower than in the case of the normal Gua: Cyt pair (7.4 × 105 nmol/min/mg/M). In summary, DNA pol R is appreciably stalled by ethylation of Gua at either N2 and O6, whereas DNA pol η is unhindered by ethylation at N2 but is stalled by ethylation at O6, proceeding at a somewhat reduced rate after successful insertion of Cyt. Steady State Kinetic Assays: The Miscoding Potentials of N2-EthylGua and O6-EthylGua. A kinetic analysis was performed to measure nucleotide discrimination frequencies by DNA pols R and η at the ethylated guanines relative to the unadducted guanine. 1. Accuracy Opposite Gua. Comparisons of the Vmax/ KM values for nucleotide misinsertion (Table 1) and mispair extension (Table 2) confirm the poor fidelity of DNA pol η, relative to DNA pol R, when copying adducted DNA templates as has been observed by others (39, 40) when copying unadducted DNA templates. The misinsertion frequencies for incorrect nucleotides by DNA pol R using the unadducted Gua template 1 predict discrimination frequencies relative to the correct nucleotide of about 1/20 000 for dGMP and dTMP and 1/120 000 for dAMP when all four dNTPs are present at equal concentration (30). The misinsertion frequencies by DNA pol η relative to the correct nucleotide are in the range of 1/330-1/740 for the three incorrect nucleotides indicating an accuracy ∼100-fold lower than that of DNA pol R. The specificity of mispair extension was similar for DNA pols R and η with the purine:purine mispairs Gua:Gua and Gua:Ade extended less efficiently than the purine:pyrimidine mispair Gua:Thy (Table 2). However, the extension frequency for the Gua:Thy mispair by DNA pol R (1/2200) is ∼200-fold greater than that of the purine: purine mispairs (1/440 000), and there is only a ∼4-fold greater extension frequency for the Gua:Thy mispair (1/ 1600) relative to the Gua:Ade mispair (1/6700) for DNA pol η. Notably, the mispair extension frequencies for DNA pol η (Table 2) are lower than the misinsertion frequencies (Table 1) suggesting that failure to extend misinserted nucleotides is a significant contribution to the overall fidelity, albeit low, for the DNA pol η. 2. Miscoding Opposite Adducts: DNA Pol r. The misinsertion frequency by DNA pol R (Table 1) using the O6-ethylGua template 3 predicts a preference of ∼2-fold for misinsertion of dTMP relative to insertion of the correct dCMP nucleotide. Indeed, this tendency for misinsertion of dTMP opposite O6-ethylGua by DNA pol R has been reported (11). Importantly, a large component of the poor nucleotide discrimination measured at the O6ethylGua lesion by DNA pol R can be attributed to a major reduction in the Vmax/KM value for insertion of the correct dCMP nucleotide (Table 1). The very large Vmax/ KM value for extension from the O6-ethylGua:Thy mispair of 2.9 × 106 nmol/min/mg/M for DNA pol R further substantiates the high miscoding potential of the O6ethylGua lesion for DNA pol R. These results indicate a miscoding potential and specificity by the O6-ethylGua lesion for DNA pol R that is similar to that of the O6methylGua lesion (34, 41).

Chem. Res. Toxicol., Vol. 16, No. 12, 2003 1621

Figure 4. Single primer extension reaction (60 µL) using the N2-ethylGua template was incubated with DNA pol R (0.6 ng/ µL), and samples (10 µL) were removed after 1 (lane 2), 5 (lane 3), and 10 min (lane 4) at 37 °C. The DNA pol η was added to 0.94 ng/µL to the remainder of the reaction (30 µL), and samples (10 µL) were removed after an additional 1 (lane 5), 5 (lane 6), and 10 min (lane 7) at 37 °C. The reaction in lane 1 contained no enzyme, and the reaction (10 µL) in lane 8 contained DNA pol η (0.94 ng/µL) at 37 °C for 10 min.

Nucleotide misinsertion frequencies opposite N2-ethylGua by DNA pol R were so low that they were below the level of detection in this assay. Extension from the N2ethylGua:Thy mispair with a Vmax/KM value of 1.1 × 104 nmol/min/mg/M by DNA pol R was detected (Table 2). 3. Miscoding Opposite Adducts: DNA Pol η. The results of experiments with DNA pol η and the O6-ethyl adduct mirror those with DNA pol R; there was a similar 3-fold preference for misinsertion of dTMP relative to dCMP. Again, a major factor in this tendency is ascribable to the measured inefficiency of dCMP insertion (Table 1). Similarly, the Vmax/KM values for extension from the O6-ethylGua:Thy mispair by DNA pol η (4.4 × 104 nmol/min/mg/M) rival the Vmax/KM value for correct nucleotide extension by this enzyme. The data in Tables 1 and 2 summarize the remarkable observation that DNA pol η exhibits “normal” fidelity in replicating past the N2-ethylG adduct, in contrast with what has been reported in the case of the E. coli DNA pol I enzyme (25). The misinsertion frequencies by DNA pol η opposite the adducted nucleotide N2-ethylGua range from 1/550-1/1600 (Table 1) and are comparable to fidelity opposite Gua. Furthermore, the extension frequencies from mispaired nucleotides positioned opposite the N2-ethylGua by DNA pol η indicate discrimination levels of 1/2700 or better, as good as those detected from mispairs at the unadducted Gua (Table 2). Polymerization of the N2-EthylGua Template by the Combined Actions of DNA Pols r and η. A primer extension experiment was performed that indicates that the DNA pol η can rescue polymerization initiated by DNA pol R that is stalled at the N2-ethylGua lesion. DNA pol η was added to a reaction containing the N2-ethylGua template after an initial incubation with DNA pol R alone (Figure 4). As expected, incubation of the N2-ethylGua template with DNA pol R for 10 min resulted in the accumulation of greater than 90% of the elongated product at the 14-mer position resulting from the blocking effect of the N2-ethylGua adduct (Figure 4, lanes

1622 Chem. Res. Toxicol., Vol. 16, No. 12, 2003

2-4). Subsequent addition of DNA pol η resulted in further polymerization of about 70% of the stalled elongation product to the end of the DNA template (Figure 4, lanes 5-7). In a control reaction, addition of DNA pol η alone resulted in only 5% of the 12-mer primer elongated past the N2-ethylGua adduct indicating that relatively low amounts of DNA pol η provide the necessary polymerization past the N2-ethylGua adduct that has blocked the DNA pol R. Finally, it is noted with respect to possible biological implications that although DNA pol η clearly does rescue DNA pol R at the N2ethylguanine, any replication by DNA pol η occurs with inherently less fidelity at the site of lesion and beyond. It is possible that interactions between the lesion bypass enzymes such as DNA pol η and other replication proteins such as PCNA (43) regulate polymerization in cells to limit synthesis to the site of the adducted nucleotide. The overall mutagenic potential for any lesion in DNA depends on its frequency for occurrence, the cell’s capacity to repair the lesion, and the miscoding potential of the lesion. The primer extension and kinetic analysis presented here suggests that the N2-ethylGua lesion in DNA might represent a strong block for the replicative DNA pols such as DNA pol R. The accurate translesion synthesis of the N2-ethylGua lesion in DNA by DNA pol η in mammalian cells would permit continued replication resulting in a relatively low mutagenic potential for the N2-ethylGua lesion. This is in contrast to O6-ethylGua in which translesion miscoding by the replicative DNA pol R readily occurs, yet translesion synthesis by the “bypass” DNA pol η is markedly impaired and highly error prone. These observations suggest that DNA pol η has little role in modulating the mutagenicity of O6ethylGua but might be crucial to the accurate polymerization past the N2-ethylGua lesion. We are currently undertaking further experiments using the N2- and O6ethylguanine lesions in vivo in order to test this idea.

Summary DNA pol R is appreciably stalled by ethylation of Gua at either N2 or O6, by factors of 103-104, whereas DNA pol η is unhindered by ethylation at N2 but is stalled by ethylation at O6. The O6-ethylGua lesion directs mispairing of dTMP over the correct dCMP by both DNA pols R and η with similar frequencies for the two enzymes. N2-ethylGua is such a powerful block to polymerization by DNA pol R that the rate of misinsertion is not measurable in this study. However, DNA pol η processes N2-ethylGua at rates, and with misinsertion frequencies, comparable to that of unadducted Gua. Thus, DNA pol η rescues DNA pol R in polymerization passed the lesion N2-ethylGua, but it does so with a fidelity that is compromised relative to the fidelity of DNA pol R.

Acknowledgment. This work was sponsored by NIH Grants CA88950 and CA12197. Supporting Information Available: Schemes and methods for the synthesis of N2-Ethyl-(N,N-diisopropyl-2-cyanoethyl)5′-(p-dimethoxytrityl)-2′-deoxyguanosine phosphoramidite and N2-phenoxyacetyl-O6-ethyl-3′-(N,N-diisopropyl-2-cyanoethyl)-5′(p-dimethoxytrityl)-2′-deoxyguanosine phosphoramidite, analytical HPLC chromatograms of the purified oligonucleotides 1

Perrino et al. and 2, and SDS-PAGE analysis of the human DNA pol η. This material is available free of charge via the Internet at http:// pubs.acs.org.

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