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J. Biol. Chem., Vol. 281, Issue 50, 38244-38256, December 15, 2006

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Translesion Synthesis across O⁶-Alkylguanine DNA Adducts by Recombinant Human DNA Polymerases^*

Jeong-Yun Choi¹, Goutam Chowdhury¹², Hong Zang², Karen C. Angel, Choua C. Vu^¶, Lisa A. Peterson^¶, and F. Peter Guengerich³

From the Department of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146, the Department of Pharmacology, College of Medicine, Ewha Womans University, Seoul 158-710, Republic of Korea, and ^¶Division of Environmental Health Sciences and the Cancer Center, University of Minnesota, Minneapolis, Minnesota 55455

Received for publication, August 31, 2006 , and in revised form, October 10, 2006.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Previous studies have shown that replicative bacterial and viral DNA polymerases are able to bypass the mutagenic lesions O⁶-methyl and -benzyl (Bz) G. Recombinant human polymerase (pol) also copied past these two lesions but was totally blocked by O⁶-[4-oxo-4-(3-pyridyl)butyl] (Pob)G, an important mutagenic lesion formed following metabolic activation of the tobacco-specific carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. The human translesion pols and produced mainly only 1-base incorporation opposite O⁶-MeG and O⁶-BzG and had very low activity in copying O⁶-PobG. Human pol copied past all three adducts. Steady-state kinetic analysis showed similar efficiencies of insertion opposite the O⁶-alkylG adducts for dCTP and dTTP with pol and ; pol showed a strong preference for dTTP. pol , , and showed pre-steady-state kinetic bursts for dCTP incorporation opposite G and O⁶-MeG but little, if any, for O⁶-BzG or O⁶-PobG. Analysis of the pol O⁶-PobG products indicated that the insertion of G was opposite the base (C) 5' of the adduct, but this product was not extended. Mass spectrometry analysis of all of the pol primer extension products indicated multiple components, mainly with C or T inserted opposite O⁶-alkylG but with no deletions in the cases of O⁶-MeG and O⁶-PobG. With pol and O⁶-BzG, products were also obtained with –1 and –2 deletions and also with A inserted (opposite O⁶-BzG). The results with pol may be relevant to some mutations previously reported with O⁶-alkylG adducts in mammalian cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Chemical and physical damage to DNA can cause mutations and ultimately cancer, cardiovascular disease, aging, and other diseases (1–4). This damage can result from endogenous agents or exogenous chemicals. Many types of damage are known, and the biological effects can vary considerably. One of the prominent types of damage is alkylation at the O-6 atom of guanine (5, 6). O⁶-AlkylG⁴ lesions are some of the more mutagenic lesions formed from DNA-alkylating agents (5, 7). The ability of O⁶-alkylG adducts to cause mutations has been demonstrated directly in site-specific mutagenesis experiments with defined adducts (8–12). Further support for the view that these are deleterious species derives from the existence of specific DNA repair systems for this type of damage in almost all species, ranging from most bacteria to humans (13, 14).

Different alkylating agents form O⁶-alkylG adducts, and structure-activity relationships are important for understanding the basic mechanisms of how DNA polymerases function as well as issues such as carcinogenesis. Studies have been done on the comparative mutagenesis of O⁶-MeG, O⁶-ethylG, and O⁶-BzG⁵ lesions in Escherichia coli (11), but in vivo experiments have caveats about the effects of DNA repair (e.g. some O⁶-alkylG DNA-alkyltransferases are considerably more proficient with O⁶-BzG than O⁶-MeG (15)) (Fig. 1). O⁶-EthylG and O⁶-BzG produce some nontargeted mutations in the H-ras gene in Rat4 cells (9). Previous work on blockage and misincorporation opposite O⁶-MeG and O⁶-BzG has been done in this laboratory with the replicative model polymerases pol T7^– and HIV-1 RT (16) and by others with several DNA polymerases, mostly bacterial (7, 17, 18). Some of the more significant conclusions with pol T7^– and HIV-1 RT were that the bulk of the adduct at the O-6 atom had an inhibitory effect and that an inactive polymerase-oligonucleotide complex is in equilibrium with the functional form (16, 19).

Studies with pol T7^– and HIV-1 RT indicated that the effect of size at the N-2 atom is more severe than at the O-6 atom (16, 19, 20). However, these issues have not been addressed in detail with mammalian DNA polymerases, particularly replicative pol and the so-called translesion polymerases that bypass damage in cells (Y family, e.g. pol , , and ). We have systematically addressed the issue of the effect of bulk at the N-2 guanine atom on the insertion of dNTPs by several of the above polymerases (21–23), and we now apply similar quantitative approaches to O⁶-alkylG adducts varying in size. The studies with O⁶-MeG and O⁶-BzG were extended to the larger adduct O⁶-PobG, which is of relevance to tobacco-induced carcinogenesis (24–28). The O⁶-PobG adduct is formed from the "tobacco-specific" carcinogen 4-(methylnitrosoamino)-1-(3-pyridyl)-1-butanone in vivo (27) and has been demonstrated to be mutagenic in both bacterial and mammalian cells (24, 25).

View larger version (6K): [in this window] [in a new window]   FIGURE 1. O6-AlkylG derivatives studied in this work.

View larger version (58K): [in this window] [in a new window]   FIGURE 2. Extension of 32P-labeled primers opposite G, O6-MeG, O6-BzG, and O6-PobG with all four dNTPs present. A, pol ; B, pol ; C, pol ; D, pol . The primer (24-mer) was annealed with each of the four different 36-mer templates (Table 1) containing an unmodified G or O6-modified G placed at the 25th position from the 3'-end. Reactions were done for 15 min with increasing concentrations of polymerases, as indicated, and 100 nM of DNA substrate (primer-template) as indicated. In the case of pol (A), 400 nM PCNA was also included (32). The 32P-labeled 24-mer primer was extended in the presence of all four dNTPs. Reaction products were analyzed by denaturing gel electrophoresis with subsequent PhosphorImaging analysis. A, the degraded bands are attributed to the inherent exonuclease activity of pol .

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—Unlabeled dNTPs were obtained from Amersham Biosciences; S_p-dCTPS was from Biolog Life Science Institute (Bremen, Germany), and [-³²P]ATP (specific activity 3 x 10³ Ci mmol^–1) was from PerkinElmer Life Sciences. T4 polynucleotide kinase and restriction endonucleases were purchased from New England Biolabs (Beverly, MA). Bio-Spin columns were obtained from Bio-Rad. A protease inhibitor mixture (mixture) was from Roche Applied Science.

Oligonucleotides—The unmodified oligonucleotides and the O⁶-MeG-modified 36-mer (Table 1) were purchased from Midland Certified Reagent Co. (Midland, TX). The 36-mer containing O⁶-BzG was prepared using a modification of the procedure described elsewhere (16).

The extinction coefficients for the oligonucleotides, estimated by the Borer method (30), were as follow: 24-mer, ₂₆₀ = 224 mM^–1 cm^–1; 25-mer, ₂₆₀ = 232 mM^–1 cm^–1; and 36-mer, ₂₆₀ = 310 mM^–1 cm^–1 (20, 21).

Expression and Purification of Human DNA Polymerases—Recombinant human pol (21, 31), pol (21), pol (22), pol (23), and PCNA (32, 33) were prepared as described previously.

Reaction Conditions for Enzyme Assays—Standard DNA polymerase reactions were done in 50 mM Tris-HCl (pH 7.5) buffer containing 5 mM DTT, 100 µg of bovine serum albumin ml^–1 (w/v), and 10% glycerol (v/v) with 100 nM primer-template at 37 °C (20–22), with 50 mM NaCl added in the experiments with pol (21). Primers were 5'-end-labeled using T4 polynucleotide kinase/[-³²P]ATP and annealed with template (36-mer). All reactions were initiated by the addition of dNTP solutions containing MgCl₂ (5 mM final concentration) to preincubated enzyme/DNA mixtures.

Primer Extension Assay with All Four dNTPs ("Run-on" or "Standing Start" Experiments)—A ³²P-labeled primer, annealed to either an unmodified or modified (O⁶-alkylG) template, was generally extended in the presence of all four dNTPs (100 µM each) for 15 min. In some cases (extension of A:O⁶-alkylG mismatches), the dNTP concentrations were raised to 1 mM, and the incubation time was 60 min. Reaction mixtures (8 µl) were quenched with 2 volumes of a solution of 20 mM EDTA in 95% formamide (v/v) (20). Products were resolved using a 16% PAGE system (w/v) containing 8 M urea and visualized using a Molecular Imager FX and Quantity One software (Bio-Rad).

Steady-state Kinetic Analyses—A ³²P-labeled primer, annealed to either an unmodified or adducted template, was extended in the presence of varying concentrations of a single dNTP. The molar ratio of primer-template complex to enzyme was at least 10:1. Polymerase concentrations and reaction times were chosen so that maximal product formation was 20% of the substrate concentration (34). The primer-template complex was extended with dNTP in the presence of 0.1–5 nM enzyme for 5–30 min. All reactions (8 µl) were done at 10 dNTP concentrations (in duplicate) and quenched with 2 volumes of a solution of 20 mM EDTA in 95% formamide (v/v) (20–22). Products were resolved using 16% polyacrylamide gels (w/v) containing 8 M urea and quantitated with PhosphorImaging analysis using a Molecular Imager FX instrument and Quantity One software (Bio-Rad). Graphs of product formation versus dNTP concentration were fit using nonlinear regression (hyperbolic fits) in GraphPad Prism version 3.0 (San Diego, CA) for the determination of k_cat and K_m values.

View larger version (15K): [in this window] [in a new window]   FIGURE 3. Pre-steady-state burst kinetics of incorporation opposite G, O6-MeG, and O6-BzG by human pols and . A, pol ; B, pol . pol or (25 and 17 nM, respectively) was incubated with 100 nM 24-mer/36-mer primer-template complex in a rapid quench-flow instrument and mixed with dCTP (and MgCl2) to initiate reactions as follows: 1 mM dCTP for the 24-mer/36-G-mer (), 24-mer/36-O6-MeG-mer (), and 24-mer/36-O6-BzG-mer (). The polymerization reactions were quenched with 0.3 M EDTA at the indicated time intervals, and product formation was determined following gel electrophoresis separation and PhosphorImaging. The data were fit to the following burst equation: y = A(1 – e–kpt) + ksst, as described under "Experimental Procedures" (without normalization of kss for enzyme concentration in the equation). Pre-steady-state rates (kp) were estimated for pol as follows: G-mer, kp = 26.3 ± 4.8 s–1 and kss = 0.95 ± 0.06 s–1; O6-MeG-mer, kp = 4.4 ± 0.7 s–1 and kss = 0.088 ± 0.006 s–1; O6-BzG-mer, kp = 1.7 ± 0.1 s–1 and kss = 0.16 ± 0.01 s–1. For pol the values were as follows: G-mer, kp = 11 ± 3 s–1 and kss = 0.85 ± 0.08 s–1; O6-MeG-mer, kp = 2.7 ± 0.4 s–1 and kss = 0.27 ± 0.01 s–1; O6-BzG-mer, kp = 0.6 ± 0.3 s–1 and kss = 0.034 ± 0.007 s–1.

View larger version (15K): [in this window] [in a new window]   FIGURE 4. Pre-steady-state kinetic analysis of incorporation of dCTP and dTTP opposite G, O6-MeG, and O6-BzG by human pol . A, dCTP incorporation; B, dTTP incorporation. pol (24 nM) was incubated with 100 nM 24-mer/36-mer primer-template complex in a rapid quench-flow instrument and mixed with 1 mM dCTP or dTTP (and MgCl2) to initiate reactions as follows: 24-mer/36-G-mer (), 24-mer/36-O6-MeG-mer (), and 24-mer/36-O6-BzG-mer (). The polymerization reactions were quenched with 0.3 M EDTA at the indicated time intervals, and product formation was determined following separation by gel electrophoresis and PhosphorImaging. The data were fit to the following burst equation: y = A(1 – e–kpt) + ksst, as described under "Experimental Procedures" (without normalization of kss for enzyme concentration in the equation). Pre-steady-state rates (kp) were estimated for dCTP as follows: G-mer, kp = 3.5 ± 0.5 s–1 and kss = 0.36 ± 0.02 s–1; O6-MeG-mer, kp = 0.9 ± 0.3 s–1 and kss = 0.25 ± 0.03 s–1; O6-BzG-mer, kp = 1.2 ± 0.2 s–1 and kss = 0.29 ± 0.01 s–1. With dTTP the values were as follows: G-mer, kp = 0.5 ± 0.1 s–1 and kss = 0.11 ± 0.01 s–1; O6-MeG-mer, kp = 1.6 ± 0.2 s–1 and kss = 0.4 ± 0.03 s–1; O6-BzG-mer, kp = 1.3 ± 0.3 s–1 and kss = 0.33 ± 0.04 s–1.

Phosphorothioate Analysis—With the ³²P-primer annealed to either an unmodified or adducted template, reactions were initiated by rapid mixing of ³²P-primer-template/polymerase mixtures (12.5 µl) with S_p-dCTPS-Mg²⁺ complex (or dCTP-Mg²⁺) (10.9 µl) and then quenched with 0.3 M EDTA after reaction times varying from 5 ms to 30 s for O⁶-PobG-containing DNA. Products were analyzed as described for the pre-steady-state reactions (see above).

LC-MS/MS Analysis of Oligonucleotide Products from pol Reactions—In a typical reaction, 1–4 nmol of DNA (primer-template) and 50–100 pmol of pol were mixed in 50 mM Tris-HCl buffer (pH 7.7) containing 2 mM DTT, 50 mM NaCl, 5 mM MgCl₂, and 100 µg of bovine serum albumin ml^–1 in a total volume of 100 µl. The reaction was initiated by adding 1 mM each of four dNTPs and incubated for 4 h at 37 °C. The reaction was terminated by removal of the excess dNTPs using a spin column (Bio-Spin 6 chromatography column, Bio-Rad). To the above filtrate (100 µl), Tris-HCl buffer (50 mM), DTT (2 mM), and UDG (6 units) were added, and the volume was made up to 200 µl (37). The reaction mixture was incubated for 5 h at 37 °C, then heated at 95 °C for 1 h in the presence of 0.5 M piperidine, and finally concentrated to dryness by lyophilization; the residue was dissolved in 100 µl of H₂O for LC-MS/MS analysis.

LC-MS/MS was performed on an Acquity ultraperformance liquid chromatography system (Waters Associates) connected to a Finnigan LTQ mass spectrometer (ThermoElectron Corp., San Jose, CA), operating in the ESI negative ion mode and using an Acquity ultraperformance liquid chromatography system BEH octadecylsilane (C₁₈) column (1.7 µm, 1.0 x 100 mm). LC conditions were as follows: buffer A contained 10 mM NH₄CH₃CO₂ plus 2% CH₃CN (v/v), and buffer B contained 10 mM NH₄CH₃CO₂ plus 95% CH₃CN (v/v). The following gradient program was used with a flow rate of 150 µl min^–1: 0–3 min, linear gradient from 100% A to 97% A/3% B (v/v); 3–4.5 min, linear gradient to 80% A/20% B (v/v); 4.5–5 min, linear gradient to 100% B; 5–5.5 min, hold at 100% B; 5.5–6.5 min, linear gradient to 100% A; 6.5–9.5 min, hold at 100% A. The temperature of the column was maintained at 50 °C. Samples (10 µl) were infused with an autosampler system. ESI conditions were as follows: source voltage 4 kV, source current 100 µA, auxiliary gas flow rate setting 20, sweep gas flow rate setting 5, sheath gas flow setting 34, capillary voltage –49 V, capillary temperature 350 °C, tube lens voltage –90 V. MS/MS conditions were as follows: normalized collision energy 35%, activation Q 0.250, and activation time 30 ms. Product ion spectra were acquired over the range m/z 300–2000. The triply (negatively) charged species were generally used for CID analysis; in some cases the –4 ions were used. The calculations of the CID fragmentations of oligonucleotide sequences were done using a program linked to the Mass Spectrometry Group of Medicinal Chemistry at the University of Utah (38).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Overall Strategy—As in previous work with the two replicative viral polymerases HIV-1 RT and pol T7^– (16, 19), comparisons were made with the four human DNA polymerases (, , , and ) with Me and Bz substituents at the O-6 atom of G. In addition, an even larger adduct of direct relevance to tobacco-induced carcinogenesis, O⁶-PobG, was included in comparisons. Initial studies involved extension of a 24-mer primer opposite templates containing each of the adducts, followed by more quantitative steady-state kinetic analysis of the insertion reactions. Further analysis involved pre-steady-state kinetic measurements of incorporation with three of the polymerases (, , and ). The most active polymerase with all three O⁶-alkylG adducts (pol ) was analyzed to characterize all the products.

Primer Extension Past O⁶-AlkylG Adducts Using All Four dNTPs—Preliminary studies indicated that pol activity is stimulated by the presence of PCNA. None of the other polymerases were stimulated by the addition of PCNA under these conditions, without or with these or any of several other DNA adducts (21–23). pol readily extended the primer opposite the O⁶-MeG and O⁶-BzG templates but was completely blocked by O⁶-PobG, and exonuclease activity was seen in the latter case (Fig. 2A). pol was hindered by all of the O⁶-alkylG adducts but nevertheless extended all three (Fig. 2B). The product derived from the primer opposite O⁶-PobG appeared not to be full-length, and the analysis is considered later in this work. pol showed considerable 1-base incorporation opposite O⁶-MeG and O⁶-BzG but little opposite O⁶-PobG (Fig. 2C). pol showed limited 1-base incorporation and extension beyond O⁶-MeG and O⁶-BzG but very little activity with O⁶-PobG (Fig. 2D).

Steady-state Kinetics of dNTP Incorporation Opposite G and O⁶-AlkylG Adducts—Preliminary analyses with individual dNTPs showed that, as with processive polymerases (16, 39), mainly C or T was incorporated opposite O⁶-MeG and O⁶-BzG. With O⁶-PobG, some dGTP incorporation also occurred, and incorporation was subsequently measured with all four dNTPs with pol . Rates of incorporation of dATP by pol were also measured for all three adducts because of results obtained later in the work (see below). Rates were measured as a function of the concentration of each dNTP, and the steady-state kinetic parameters were determined (Table 2).

View this table: [in this window] [in a new window]   TABLE 2 Steady-state kinetic parameters for 1-base incorporation by human pol , , , and

View larger version (14K): [in this window] [in a new window]   FIGURE 5. Phosphorothioate analysis of pre-steady-state kinetics of dCTP incorporation opposite O6-PobG by human pol . pol (50 nM) was incubated with 100 nM 24-mer/36-mer primer-template complexes (32P-labeled primer) in a rapid quench-flow instrument and mixed with 1 mM dCTP () or Sp-dCTPS () to initiate reactions for the 24-mer/36-O6-PobG-mer. The following rates were estimated: dCTP, kp = 0.22 ± 0.03 s–1; Sp-dCTPS, kp = 0.14 ± 0.05 s–1.

View larger version (28K): [in this window] [in a new window]   FIGURE 6. Extension of primers with C, T, or G positioned opposite O6-PobG by pol . Primer (24-mer, Table 1, with an additional C, T, or G at the 3'-end) was annealed to 36-mer template (Table 1) containing an O6-PobG placed at the 25th position from the 3'-end. Reactions were done for 15 min with increasing concentrations of pol , as indicated, and 100 nM of DNA substrate (primer-template) as indicated. The 32P-labeled primers were extended in the presence of all four dNTPs. Reaction products were analyzed by denaturing gel electrophoresis with subsequent PhosphorImaging analysis.

With O⁶-PobG in the template, dGTP was also incorporated. The order of preference of individual dNTPs varied among the polymerases, showing the order (based on k_cat/K_m) C > G > A > T for pol , T > G > C for pol , and C G > T for pol . The k_cat/K_m for dCTP incorporation opposite O⁶-PobG by pol is considerably less than for O⁶-MeG or O⁶-BzG by the same enzyme but still much higher than for incorporation of any dNTPs opposite this adduct by any of the other polymerases.

View larger version (29K): [in this window] [in a new window]   FIGURE 7. Products of extension of O6-alkylG template-primer complexes by pol . Data used to derive these results are presented in Figs. 8, 9, 10 and Tables 3, 4, 5 and in the Supplemental Material (supplemental Figs. S3–S13 and Tables S1–S8).

View larger version (27K): [in this window] [in a new window]   FIGURE 8. LC-MS/MS analysis of the pol–1 deletion product of extension of the primer opposite the template containing O6-BzG. The reaction mixture containing the oligonucleotide fragments obtained by UDG hydrolysis. A, total ion current trace of the extension products. B, ESI mass spectrum of the peaks eluted at 3.25 min in A. C, reconstructed single reaction monitoring profile of m/z 1027.2. D, CID spectrum of m/z 1027.2. See Table 3 for lists of assignments of CID ions. The m/z 945.0 and 1260.1 ions are the –4 and –3 charged species, respectively, resulting from the same product (–2 deletion product; supplemental Fig. S10); the m/z 1027.2 and 1370.1 ions are the –4 and –3 charged species, respectively, resulting from another product, namely the one used for the CID (D) and shown with the sequence at the top of the figure.

As expected from the results of studies with other oligonucleotide sequences, kinetic bursts were observed with pol , , and for insertion of dCTP opposite G (Figs. 3 and 4) (21–23). These results indicate that a step in catalysis occurring after product formation is at least partially rate-limiting (42). The bursts seen with pol (Fig. 4) do not break as sharply as with pol and pol (Fig. 3), probably because of the slower k_p. (The amount of pol available was not sufficient to conduct these experiments.)

Some burst character was retained when the experiments were repeated with O⁶-MeG in the cases of pol and (Fig. 3). With O⁶-BzG the bursts were largely abolished. The loss of the burst is interpreted to mean that a step occurring prior to the formation of product (presumably phosphodiester bond formation or possibly a conformational step) is now slower than product release. With pol (Fig. 4), the trend in the kinetics was G > O⁶-BzG > O⁶-MeG (Fig. 4A), which was unexpected. An opposite pattern was seen for dTTP incorporation with pol , which is very error-prone with these lesions (Table 1), and only a semblance of a burst was observed with both O⁶-MeG and O⁶-BzG (Fig. 4B).

Phosphorothioate Analysis of dCTP Incorporation Opposite O⁶-PobG by pol —One approach to further delineate aspects of catalysis by DNA polymerases is the use of sulfur versus oxygen elemental ("thio") effects on rates. The principle is similar to that utilized with kinetic isotope effects, in that the substitution of an -oxygen of a dNTP with sulfur decreases the electronegativity and makes bond breakage more difficult. If this is a slow step in the reaction, then the overall reaction will be slowed (43, 44). This is a simplistic interpretation and, as with many aspects of kinetic hydrogen isotope effects, the detailed interpretation of the extent of the changes has some differences in opinion (43, 45, 46).

pol was studied in the context of an elemental effect for incorporation of dCTP opposite O⁶-PobG, in that this is the most active of the polymerases studied here with this adduct. The elemental effect on the early incorporation phase was only 1.5 (Fig. 5), only slightly higher than reported for incorporation of dCTP opposite G with this enzyme (21).

pol Extension beyond O⁶-AlkylG Paired with Bases—Of the human DNA polymerases examined, pol and pol were the most catalytically efficient in incorporating dNTPs opposite the O⁶-MeG and O⁶-BzG adducts (Table 2). pol was the only polymerase that could incorporate opposite O⁶-PobG with appreciable efficiency (Table 2) and extend the product (Fig. 2). pol inserted C, T, and G (Table 2), but this information by itself may not predict the outcome of subsequent extension steps. To address the issue, we prepared primers with either C, T, or G positioned opposite O⁶-PobG and used varying concentrations of pol in the presence of all four dNTPs (Fig. 6). The primers with the pyrimidines (C, T) positioned opposite O⁶-PobG were extended; with G the extension was much slower. The results suggest that when G is inserted, it is positioned opposite the C 5' of the O⁶-PobG in the sequence and that the resulting distortion is not favorable for extension by pol .

In the work with pol , we also detected some incorporation of A opposite each of the three O⁶-alkylG adducts (Table 2). The catalytic efficiency was low and roughly similar with each adduct. To determine whether these products (with A positioned opposite O⁶-alkylG adducts) are extended, we prepared the (radiolabeled) primers with the A at the 3' position and incubated with varying concentrations of pol in the presence of all four dNTPs. The results showed very limited extension of primers having an A paired with any of the three O⁶-alkylG lesions studied here, particularly O⁶-PobG (supplemental Fig. S2).

Analysis of Primer Incorporation/Extension Products Using LC-MS/MS with pol Extension Products—To understand misincorporation and mutational events, it is desirable to determine the effects of modified DNA templates on the outcome of multiple primer incorporation events, which have not been specifically addressed in the experiments presented thus far in this paper. Many of the events involve incorporation of a single dNTP (Table 2 and Figs. 3, 4, 5) or multiple incorporation (extension events in which the products are not characterized) (Figs. 2 and 6). We utilized an approach to the sequence analysis of oligonucleotides using LC-MS/MS (47), which has been modified for this work. A key element for the success of this method is the placement of uracil residues in the primer strand and the use of UDG to then reduce the size of the product to facilitate sequence analysis with LC-MS/MS (37).

The analysis of the extension products of the primer opposite the O⁶-alkylG lesions presented a technical challenge, in that complex mixtures of products were obtained even when a high concentration of pol was used (50 pmol (not shown), cf. 4–10 pmol in Figs. 2 and 6), and the concentration of dNTPs was raised to 1 mM each in these assays. pol has a tendency to stop just short of the terminus of the primer-template complex, at least part of the time, but can also add residues at the end in "blunt-end" additions. Thus, discerning whether the shorter lengths of products is because of frameshifts or to stopping at the end of the primer was not possible without sequence analysis.

The results of the pol experiments with the three templates are shown in Fig. 7. For O⁶-MeG and O⁶-PobG, all of the detected products resulted from insertion of C (correct) or T opposite the modified G. With O⁶-MeG, 77% of the products resulted from T insertion (23% C), and with O⁶-PobG the combined T insertion products accounted for 22% of the products (78% C). The lengths of the products of the O⁶-MeG products, and to some extent the O⁶-PobG products, were slightly shorter than in the case of the O⁶-BzG products (Fig. 7) (in apparent contrast to the gel results shown in Fig. 2, although the conditions used in the experiments in Fig. 2 were not as prolonged). None of the products contained a G inserted opposite the O⁶-PobG.

View larger version (21K): [in this window] [in a new window]   FIGURE 9. LC-MS/MS analysis of the pol T insertion product of extension of the primer opposite the template containing O6-BzG. The reaction mixture containing the oligonucleotide fragments obtained by UDG hydrolysis. The total ion current trace is shown in Fig. 8A. A, ESI mass spectrum of the peaks eluted at 4.19 min in Fig. 8A. B, reconstructed single reaction monitoring profile of m/z 1103.3. C, CID spectrum of m/z 1103.3. See Table 4 for lists of assignments of CID ions.

LC-MS/MS analysis indicated the presence of eight products (Fig. 8). Initial LC showed a complex mixture including proteins and reagents with the oligonucleotides eluting at t_R 4 min (with an additional oligonucleotide subsequently found at t_R 3.25 min) (Fig. 8A). The mass spectra showed seven peaks at m/z 945.0, 1027.2, 1099.6, 1103.3, 1105.5, 1177.8, and 1181.9, which are apparently the –4 charge ions (Fig. 8B) (subsequent work showed that the m/z 1181.9 peak was because of two separate products; see supplemental Fig. S11). A reconstructed trace of the m/z 1027.2 ion is shown in Fig. 8C. CID of the m/z 1027.2 ion (product eluted at t_R 3.25 min) produced the fragments shown in Fig. 8D. Inspection of these with the sequences of possible products (38) led to the assignment of the sequence as that shown at the top of Fig. 8 (Table 3) with a –1 deletion following O⁶-BzG, followed then by error-free polymerization until the end (yielding a net –1 frameshift) (Figs. 7 and 8). Integration of the reconstructed ion chromatograms for the –4 ions led to the assignment of relative yields for the products identified in Figs. 7 and 8B, assuming equal ionization, and the products were identified using CID analysis (Figs. 7, 8, 9, 10; supplemental Figs. S3–S13; Tables 3, 4, 5; and supplemental Tables S1–S8). The main product (37%) resulted from error-free polymerization. Two products with T inserted opposite O⁶-BzG were obtained (total of 31%), with one including a blunt-end A addition at the end (Fig. 9). Another product, with A inserted opposite O⁶-BzG, accounted for 11% of the total product (Fig. 10). Accurate polymerization occurred after insertion of T; one product stopped 1 base short of the end and the other (minor) 2 bases short (Fig. 9).

View this table: [in this window] [in a new window]   TABLE 3 Observed and theoretical CID fragmentation for pol – 1 deletion product with O6-BzG The assigned sequence was 5'-pAGGAGGACGCCGA-3' (Fig. 8). See Ref. 53 for nomenclature of fragments.

View this table: [in this window] [in a new window]   TABLE 4 Observed and theoretical CID fragmentation for pol T insertion product with O6-BzG The assigned sequence was 5'-pAGTGAGGACGCCGA-3' (Fig. 9). See Ref. 53 for nomenclature of fragments.

View this table: [in this window] [in a new window]   TABLE 5 Observed and theoretical MS fragmentation for pol A misinsertion product with O6-BzG The assigned sequence was 5'-pAGAGAGGACGCCGA-3' (Fig. 10). See Ref. 53 for nomenclature of fragments.

View larger version (22K): [in this window] [in a new window]   FIGURE 10. LC-MS/MS analysis of the pol A misinsertion product of extension of the primer opposite the template containing O6-BzG. The reaction mixture containing the oligonucleotide fragments obtained by UDG hydrolysis. The total ion current trace is shown in Fig. 8A. A, ESI mass spectrum of the peaks eluted at 4.12 min in Fig. 8A. B, reconstructed single reaction monitoring profile of m/z 1105.5. C, CID spectrum of m/z 1105.5. See Table 5 for lists of assignments of CID ions.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Several of the major DNA polymerases were examined, using recombinant preparations. Of these, only pol has shown a requirement for the presence of PCNA in copying past DNA adducts on relatively short oligonucleotides (32, 56); pol copied reasonably well past O⁶-MeG and O⁶-BzG but was completely blocked by O⁶-PobG (Fig. 2). The translesion polymerases pol and could insert C and T opposite the O⁶-alkylG adducts but, except in the case of pol with O⁶-MeG, were not very efficient in extending the initial product.

pol was the most efficient of these enzymes in processing all three of the O⁶-alkylG residues (Fig. 1 and Table 2). Both the steady-state kinetic estimates of catalytic efficiency (single-base incorporation) and LC-MS/MS sequence analyses of the extended products show incorporation of C opposite the O⁶-alkylG adducts, with a substantial incorporation of T (Table 2 and Fig. 9). G could be inserted opposite O⁶-PobG but not efficiently extended (Table 2 and Fig. 6). In the case of O⁶-BzG, but not with O⁶-MeG or O⁶-PobG, the LC-MS/MS analysis also revealed a product with A inserted (opposite O⁶-BzG) and then accurately extended and small amounts of –1 and –2 deletion products (Fig. 7). Under the conditions used here, the LC-MS/MS analysis (Fig. 7) was more sensitive in detecting minor events, e.g. incorporation and extension of A opposite O⁶-BzG, than were the ³²P-gel assays (Table 2 and supplemental Fig. S2).

View larger version (23K): [in this window] [in a new window]   FIGURE 11. Catalytic efficiency (kcat/Km) for dCTP incorporation opposite O6-alkylG adducts by human polymerases as a function of the calculated volume of the adduct at the guanine O-6 atom. Molecular volumes (Å3) of adducts at the guanine O-6 atom were calculated using the program Chem3D (version 7.0), based on the Connolly surface algorithm (57), and plotted against kcat/Km values (Table 2) for dCTP () and dTTP () incorporation for various O6-alkylG adducts by the four human polymerases. A, pol (PCNA complex); B, pol ; C, pol ; D, pol .

In the analysis of the effect of O⁶-PobG in the log (k_cat/K_m) versus size relationship (Fig. 11), the size effect (discounting any other contributors) on C and T incorporation follows a pattern for pol but is much more severe for pol . With pol there is a "linear" drop in the efficiency of dCTP incorporation but a more precipitous drop in the efficiency of dTTP incorporation. The overall result is an apparently higher fidelity index (lower misincorporation frequency, f) for misincorporation opposite an O⁶-PobG adduct compared with O⁶-MeG and O⁶-BzG (Fig. 11 and Table 2). The LC-MS/MS analysis of primer extension products (Fig. 7) also showed a higher frequency of correct insertion products for O⁶-PobG than O⁶-MeG or O⁶-BzG.

The size effects (Fig. 11) can be compared with those reported with N²-alkylG and N²-aralkylG adducts with these polymerases (21–23). With pol , the effect of substitution at the O-6 atom is considerably more pronounced than that seen at N-2 (21). With the N²-alkylG and N²-aralkylG adducts, the attenuating effect was very limited up to the large N²-methyl(anthracenyl)G adduct, and the efficiency of insertion of the preferred "wrong" base (A) was also not changed (21). With the O⁶-alkylG derivatives (Fig. 11), the addition of a Me group lowered the catalytic efficiency of C incorporation by an order of magnitude, and the efficiency was further lowered by the substitution of a Bz group. However, the efficiency of T incorporation by pol was increased by the addition of a methyl group at the O-6 position and then decreased by the substitution of larger groups (Fig. 11). With pol the efficiency of dCTP incorporation was decreased to a similar extent by a Me or a Bz group and then very strongly decreased for O⁶-PobG (Fig. 11). This effect can be compared with the N²-alkylG results (22) where a very strong decrease in the efficiency of dCTP incorporation was seen with a methyl group (and the effects of Me and Bz groups were similar). With pol the efficiency of dCTP incorporation was rather insensitive to the size of substitution at the N-2 atom. Furthermore, the tendency for misincorporation (of dTTP or dGTP) varied little with size (23). However, substitution of the O-6 atom in the present study had a very strong effect on the efficiency of incorporation by pol (Fig. 11D).

These results and several thermodynamic studies with modified oligonucleotides in the absence of polymerases (58) suggest that certain base pairing schemes may be operative with O⁶-alkylG residues, including a 2-hydrogen bond O⁶-MeG:T pair and either a wobble or protonated O⁶-MeG:C pair, based on structures of duplex DNA (in the absence of polymerases) determined by x-ray diffraction (58, 59) (Fig. 12). T_m studies show a more favorable free energy for an O⁶-MeG:C pair than an O⁶-MeG:T pair (58, 60). The thermodynamics of pairing T (dTTP) with O⁶-MeG are more favorable than for pairing T with G and are consistent with the increase in k_cat/K_m values seen here with all polymerases in going from G to O⁶-MeG (Fig. 11). However, the thermodynamics (60) alone cannot explain why the polymerases incorporate dTTP as efficiently or more efficiently than dCTP; this appears to be a kinetic effect imparted by the polymerases.

View larger version (14K): [in this window] [in a new window]   FIGURE 12. Possible structures of O6-MeG base pairs. A, O6-MeG:T mispair. B, O6-MeG:C wobble pair. C, protonated O6-MeG:C+ pair.

View larger version (11K): [in this window] [in a new window]   FIGURE 13. General kinetic mechanism for DNA polymerization. Individual steps are numbered and discussed in the text. See references (41, 44) and modifications (19, 61). E indicates pol; Dn indicates DNA substrate; E* indicates conformationally altered polymerase; E indicates nonproductive conformation of polymerase; Dn+1 indicates DNA extended by 1 base, and PPi indicates pyrophosphate.

In all of the cases examined, the absence of sharp kinetic bursts seen with incorporation (of dCTP) opposite the O⁶-alkylG adducts implies that steps associated with or occurring prior to product formation (Fig. 13) are rate-limiting. What is not clear is whether the alkylation causes the step involving formation of the phosphodiester bond to become rate-limiting (step 4 in Fig. 13) or whether a conformational or other step preceding the bond formation step (e.g. step 3) is rate-limiting. The absence of sharp bursts in the work with the O⁶-MeG and O⁶-BzG adducts and Y family DNA polymerases (Figs. 3 and 4) contrasts with the results of studies with the model polymerases HIV-1 RT and pol T7^–, where relatively sharp bursts were seen, but the extent of the burst phase was reduced (16, 19). The results of those studies were interpreted in the context of a kinetic model in which a fraction of the polymerase is in a nonproductive complex(es) in equilibrium with the productive complex (19), which is generally compatible with a recent new model for action of replicative DNA polymerases (64). In comparing the O⁶-alkylG burst analyses for the Y family polymerases with the N²-alkylG adducts, relatively sharp bursts were observed with pol and , even with considerable bulk (21, 23). With pol , the burst phase was lost with adducts larger than N²-ethylG (22).

Our misincorporation results with individual DNA polymerases may be compared with mutagenesis studies in the literature. The literature on O⁶-alkylG mutations is dominated by reports of G to A transitions, i.e. the result of incorporation of T opposite the adduct (8). However, limited information is available about which DNA polymerases are involved in O⁶-alkylG mutagenesis, both in prokaryotic and eukaryotic systems. Also, only very limited mutagenesis studies have been done with O⁶-PobG to date (24). In the present study we showed an efficiency of bypass of O⁶-PobG in the order pol > pol , , and , but in any cell the relative roles will depend upon the amounts of these (and any other relevant) DNA polymerases. Although the pol -catalyzed incorporations opposite O⁶-PobG are relatively inefficient (Table 2), they may account for cellular mutations (24); alternatively, other DNA polymerases that we have not included in this study are more involved. All of the DNA polymerases examined here did insert T opposite all of the O⁶-alkylG lesions, as expected (Table 2 and Fig. 7). Of these human DNA polymerases, all showed similar efficiencies of insertion of C and T, with two exceptions as follows: (i) pol showed a preference of an order of magnitude in inserting T > C, and (ii) with pol and k (although the efficiencies are lower) the fidelity is actually more favorable (for insertion of C) opposite O⁶-PobG than O⁶-MeG or O⁶-BzG (Table 2). With pol , analysis of the products of primer extension opposite O⁶-BzG produced five separate results as follows: (i) error-free incorporation (55%; Fig. 7); (ii) insertion of T opposite O⁶-alkylG (31%) (Figs. 7 and 9 and Table 4); (iii) a 1-base deletion (2%) (Figs. 7 and 8 and Table 3); (iv) a 2-base deletion (1%) (Fig. 7 and supplemental Fig. S10); and (v) misinsertion of A opposite O⁶-BzG (11%) (Figs. 7 and 10 and Table 5). The latter three events were not seen in analysis of the products obtained in copying O⁶-MeG and O⁶-PobG. The products varied in the extent to which pol finished elongation even after prolonged incubation (Fig. 7), but these results are not surprising in the context of the results observed with pol and unmodified oligonucleotides (Fig. 2).

The results of the primer extension opposite the O⁶-alkylG residues are of interest in the context of some of the mutation results other than T insertion (G to A transitions) that have been reported in cellular systems. In E. coli, O⁶-BzG was less miscoding than O⁶-MeG (11), consistent with our LC-MS/MS results (Fig. 7). Our own O⁶-PobG results (only C and T insertions opposite O⁶-PobG) are consistent with mutation studies in E. coli but do not explain the more complex mutations (deletions and remote mutations, 5% total) reported in human embryonic kidney 293 cells (24). However, O⁶-BzG produced more complex mutations than O⁶-MeG in an H-ras sequence in Rat4 cells (9), and the insertions of T and A opposite O⁶-BzG were seen in our LC-MS/MS work (Fig. 7). We observed deletions in the LC-MS/MS work with pol and O⁶-BzG but, in contrast to the cellular studies of Bishop et al. (9), no nontargeted (base pair) mutations at sites adjoining the adduct. Two outstanding issues are as follows: (i) effects of sequence context on the misincorporations cannot be excluded, and (ii) other (translesion) DNA polymerases that were not examined here may be responsible for some of the events.

In summary, replication by human Y family polymerases past a set of O⁶-alkylG adducts yielded patterns of results that were very distinguishable from those reported previously with N²-alkylG adducts (21, 23, 50). With all of the individual polymerases, O⁶-MeG enhanced the efficiency of dTTP incorporation, as opposed to only decreasing dCTP incorporation (Fig. 11). Of these polymerases, pol was the most efficient. pol was completely blocked by O⁶-PobG, but pol could incorporate opposite this and extend the product. Analysis of the product showed predominantly the expected incorporations of C and T in all cases, but unexpected products were an insertion of A, a –1 deletion, and a –2 deletion in the cases of O⁶-BzG adduct, which was not seen with the O⁶-MeG and O⁶-PobG adducts. The results show the intrinsic role of human pol with these adducts and the role of adduct size at the guanine O-6 atom. This work also demonstrates the complexity of products that can be generated with a single DNA polymerase (Fig. 7).

	FOOTNOTES

 
^* This work was supported in part by United States Public Health Service Grants R01 CA059887, R01 CA115309 (to L. A. P.), R01 ES010375, and P30 ES000267 (to F. P. G.). The costs of publication of this articl