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Originally published In Press as doi:10.1074/jbc.M605699200 on July 10, 2006

J. Biol. Chem., Vol. 281, Issue 35, 25297-25306, September 1, 2006
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Biochemical Basis of Genotoxicity of Heterocyclic Arylamine Food Mutagens

HUMAN DNA POLYMERASE {eta} SELECTIVELY PRODUCES A TWO-BASE DELETION IN COPYING THE N2-GUANYL ADDUCT OF 2-AMINO-3-METHYLIMIDAZO[4,5-f]QUINOLINE BUT NOT THE C8 ADDUCT AT THE NarI G3 SITE*Formula

Jeong-Yun Choi{ddagger}§, James S. Stover§||, Karen C. Angel{ddagger}§, Goutam Chowdhury{ddagger}§1, Carmelo J. Rizzo§||, and F. Peter Guengerich{ddagger}§2

From the Departments of {ddagger}Biochemistry and ||Chemistry and the §Center in Molecular Toxicology, Vanderbilt University, Nashville, Tennessee 37232-0146 and the Department of Pharmacology, Ewha Womans University, Seoul 158-710, Republic of Korea

Received for publication, June 14, 2006 , and in revised form, July 7, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Heterocyclic arylamines are highly mutagenic and cause tumors in animal models. The mutagenicity is attributed to the C8- and N2-G adducts, the latter of which accumulates due to slower repair. The C8- and N 2-G adducts derived from 2-amino-3-methylimidazo[4,5-f]quinoline (IQ) were placed at the G1 and G3 sites of the NarI sequence, in which the G3 site is an established hot spot for frameshift mutation with the model arylamine derivative 2-acetylaminofluorene but G1 is not. Human DNA polymerase (pol) {eta} extended primers beyond template G-IQ adducts better than did pol {kappa} and much better than pol {iota} or {delta}. In 1-base incorporation studies, pol {eta} inserted C and A, pol {iota} inserted T, and pol {kappa} inserted G. Steady-state kinetic parameters were measured for these dNTPs opposite the C8- and N 2-IQ adducts at both sites, being most favorable for pol {eta}. Mass spectrometry of pol {eta} extension products revealed a single major product in each of four cases; with the G1 and G3 C8-IQ adducts, incorporation was largely error-free. With the G3 N 2-IQ adduct, a –2 deletion occurred at the site of the adduct. With the G1 N 2-IQ adduct, the product was error-free at the site opposite the base and then stalled. Thus, the pol {eta} products yielded frame-shifts with the N 2 but not the C8 IQ adducts. We show a role for pol {eta} and the complexity of different chemical adducts of IQ, DNA position, and DNA polymerases.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The modification of DNA by chemical and physical agents is a common process and leads to loss of genetic stability (1), a factor in aging, cancer, and various other maladies, e.g. cardiovascular disease (2). Therefore, questions about the chemistry of the damage and the biochemical processing are of particular interest regarding many diseases, as well as basic molecular genetics (1). Repair of DNA damage is one issue, but another issue is the replication of damaged DNA and the introduction of mutations (1, 3). The field has become more complex with the discovery of a plethora of translesion DNA polymerases, some of which appear to have selective functions in replicating damaged DNA (1, 4). Today at least 14 human DNA polymerases are known (1, 5), and defining roles of individual polymerases in complex processes is an area of considerable interest (3, 5, 6). A major general issue is the elucidation of the bases for the selectivity of chemical and physical agents in producing various mutation spectra in individual genes, ultimately a factor in biological outcomes. Several contributors are as follows: (i) reaction of the chemical or physical agent with DNA; (ii) further nonenzymatic reactions of the DNA adduct; (iii) DNA repair (or lack of repair); (iv) the action of DNA polymerases; and (v) the biological effect of a particular mutation in changing the phenotype (3, 7).

Heterocyclic amines are an important class of chemical carcinogens (8–10). A number of these compounds are produced by pyrolysis of food and tobacco (10–12) and were originally identified on the basis of their extremely strong mutagenic properties in bacteria (8, 13). The heterocyclic amines have chemical (and biochemical) similarity to arylamines, several of which are recognized human carcinogens (14, 15). Several heterocyclic amines have been shown to be carcinogenic in rodents and non-human primates (16).

Arylamines and the derivative arylacetamides can show strong sequence selectivity in mutational assays. For example, in bacterial systems on adducts derived from AAF,3 a strong –2 frameshift (deletion) is seen at the "G3" site of the NarI sequence (GGCGCC), leading to the loss of a GC doublet (17–19). This phenomenon, which is not observed with the closely related compound AF, has been studied at several structural and biological levels (20–22). The basis is more complex than alteration of the DNA structure, in that not only the site within the DNA but also the cellular system influences the biological outcome (23). The NarI "hot spot" was first detected in analysis of the mutation spectrum or AAF (17); repetitive sequences have been detected as being prone to frameshift mutagenesis with various systems (18, 19). The position of the adduct within the repetitive NarI sequence has a strong effect on whether mutations occur or not, as shown with AAF (19).


Figure 1
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  		FIGURE 1.
Guanine N2- and C8 adducts of IQ.

 
One of the critical issues in the study of the biochemical mechanisms of processing of DNA damage is the ability to prepare specific chemical reagents, particularly DNA adducts. Using methods developed in our laboratories, we have been able to avoid producing mixtures of heterocyclic amine DNA adducts with the use of strategies for synthesis of appropriately modified monomers (24–26). With this strategy, we were able to synthesize both of the major DNA adducts formed by IQ, the N 2- and C8-guanyl adducts (Fig. 1). Both of these adducts are formed upon reaction of DNA with N-hydroxy IQ (27), but the proportion of the N2-IQ adduct increases with time in vivo (in rodents) due to much slower repair (27, 28). Both adducts were positioned at both the G1 and the G3 sites in the NarI sequence, which are known to be resistant and sensitive, respectively, to frameshift mutations with AAF (19, 29). The modified oligonucleotides were used in studies with purified recombinant human DNA polymerases, including the replicative pol {delta}-PCNA complex and the major translesion polymerases pol {eta}, {iota}, and {kappa}. Analysis included primer extension in the presence of all four dNTPs, steady-state kinetic analysis of single dNTP insertion, and LC-MS/MS sequencing analysis of the full-length extension products. The results show that pol {delta} is completely blocked by the IQ adducts and that pol {eta} is the most efficient of the translesion DNA polymerases in copying. pol {eta} exhibited different behavior with both of the two IQ adducts at each of the two internal NarI sites. In particular, the N2-IQ adduct (with pol {eta}) yielded only the –2 deletion product at the G3 site, which was not observed with the C8-IQ adduct. The selectivity of pol {eta} for the site-specific lesions is striking and correlates not only to the NarI G3 frameshift seen in bacteria but also a prominent GC deletion seen in several tissues of rats treated with IQ (30).


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Enzymes—Recombinant human pol {delta} (4-subunit mixture) (31), pol {eta} (31), pol {iota} (32), and pol {kappa} (33) were (individually) expressed in baculovirus-infected insect cell systems and purified as described previously. Recombinant human PCNA was prepared in Escherichia coli and purified as described elsewhere (34, 35).

Oligonucleotide Synthesis—The adducted oligonucleotides (24–26) were synthesized on an Expedite 8909 DNA synthesizer (PerSeptive Biosystems) on a 1-µmol scale using the UltraMild line of phosphoramidites (phenoxyacetyl-protected dA, 4-isopropyl-phenoxyacetyl-protected dG, acetylprotected dC, and dT phosphoramidites) and solid supports from Glen Research (Sterling, VA) (Table 1). The manufacturer's standard synthesis protocol was followed except at the incorporation of the modified phosphoramidites, which was accomplished manually, off-line. At this point, the column was removed from the instrument and sealed with two syringes, one of which contained 250–300 µl of the manufacturer's 1H-tetrazole activator solution (1.9–4.0% in CH3CN, w/v) and the other contained 250 µl of the phosphoramidite (15 mg, 98 mM in anhydrous CH2Cl2). The 1H-tetrazole and the phosphoramidite solutions were sequentially drawn through the column (1H-tetrazole first), and this procedure was repeated periodically over 30 min. After this time, the column was washed with manufacturer's grade anhydrous CH3CN and returned to the instrument for the capping, oxidation, and detritylation steps. The remainder of the synthesis was carried out in the usual manner.


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  		TABLE 1
Oligonucleotides used in this study

G* indicates G, C8-IQ G, or N2-IQ G.

 
HPLC—HPLC analyses and purifications were performed on a Beckman HPLC system with a UV diode array detector (model 166) monitored at 254 nm and utilizing 32 Karat software (version 3.1). The oligonucleotides were purified by using a 20 mM sodium phosphate buffer (pH 7.0, solvent 1) and CH3OH (solvent 2). The solvent gradient was as follows: initially 99% solvent 1 (v/v), then a 27.5-min linear gradient to 35% solvent 1 (v/v); a 2.5-min linear gradient to 50% solvent 1 (v/v) followed by 5-min isocratic at 50% solvent 1 (v/v), and then a 5-min linear gradient to initial conditions.

CGE—Electrophoretic analyses were carried out using a Beckman P/ACE Instrument System 5500 Series system, monitored at 260 nm. The P/ACE MDQ instrument used a 31.2 cm x 100 µm eCAP capillary with samples applied at 10 kV and run at 9 kV. The column was packed with the manufacturer's 100-R gel (for single-stranded DNA) using a Tris borate buffer system containing 7.0 M urea. CGE electrophoretograms of all modified oligonucleotides are presented in supplemental Figs. S1–S4.


Figure 2
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  		FIGURE 2.
Extension of 32P-labeled primer paired with a 27-mer template containing G or C8-IQ G. A, 32P-labeled 22-mer was annealed with a 27-mer containing G or C8-IQ G at position 23 (G1). B, 32P-labeled 19-mer was annealed with a 27-mer containing G or C8-IQ G at position 20 (G3). All incubations were done for 15 min with 100 nM primer-template and the indicated concentration of the polymerase in the presence of all four dNTPs. PCNA (400 nM) was present in the case of pol {delta}. The lengths of the bands are indicated.

 


Figure 3
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  		FIGURE 3.
Extension of 2P-labeled primer paired with a 27-mer template containing G or N2-IQ G. A, 32P-labeled 22-mer was annealed with a 27-mer containing G or N2-IQ G at position 23 (G1). B, 32P-labeled 19-mer was annealed with a 27-mer containing G or N2-IQ G at position 20 (G3). All incubations were done for 15 min with 100 nM primer-template and the indicated concentration of the polymerase in the presence of all four dNTPs. PCNA (400 nM) was present in the case of pol {delta}. The lengths of the bands are indicated.

 
MALDI-TOF Sequencing of Oligonucleotides—MALDI-TOF mass spectra of the four intact IG-containing templates are presented in supplemental Figs. S1–S4.

MALDI-TOF MS sequencing of modified oligonucleotides (36) was performed by treatment of purified oligonucleotides (0.3 A260 units, based on 1-ml volume) in an ammonium hydrogen citrate buffer (pH 9.4) with 2 milliunits of phosphodiesterase I. Aliquots of 4 µl were taken before enzyme addition and at 1-, 8-, 18-, 28-, and 38-min time points and added sequentially to the same vial, which was kept frozen on dry ice. A second DNA aliquot (0.3 A260 units in 24 µl of 20 mM ammonium acetate (pH 6.6)) was treated with 2 milliunits of phosphodiesterase II, and aliquots of 4 µl were taken in the same manner. The two digest mixtures were desalted using Millipore C18 Ziptips and eluted directly onto a MALDI plate in 3-hydroxypicolinic acid-containing ammonium hydrogen citrate (7 mg ml–1). MALDI-TOF analysis allowed sequential identification of nucleotides from the 3' end of the sequence for the phosphodiesterase I-treated sample and from the 5' end for the phosphodiesterase II-treated sample. Both phosphodiesterase treatments confirmed the expected positions of the G1 and G3 site adducts in the synthetic templates containing C8- and N2-IQ (data not presented).

Reaction Conditions for Enzyme Assays—Unless indicated otherwise, standard DNA polymerase reactions were done in 50 mM Tris-HCl buffer (pH 7.5) containing 5 mM dithiothreitol, 100 µg of bovine serum albumin ml–1 (w/v), and 10% glycerol (v/v) with 100 nM primer-template at 37 °C. Primers (19- or 22-mer) were 5' end-labeled using T4 polynucleotide kinase/[{gamma}-32P]ATP and annealed with templates (27-mers). 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 (N 2-IQ G or C8-IQ G) template, was extended in the presence of all four dNTPs (100 µM each) for 15 min. Reaction mixtures (8 µl) were quenched with 10 volumes of a solution of 20 mM EDTA in 95% formamide (v/v). Products were resolved using a 16% (w/v) PAGE system containing 8 M urea and visualized using a Molecular Imager (model 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, except for the adducted templates with pol {kappa} and pol {iota} (molar ratio of 5–30:1). Polymerase concentrations and reaction times were chosen so that maximal product formation was ≤20% of the substrate concentration. The reactions with the modified oligonucleotides were relatively slow, as shown with the results (which are typical for studies in this field). The primer-template complex was extended with each dNTP in the presence of 0.1–20 nM enzyme for 10 min. All reactions (8 µl, done at 10 dNTP concentrations) were quenched with 10 volumes of a solution of 20 mM EDTA in 95% formamide (v/v). Products were resolved using a 16% polyacrylamide (w/v) electrophoresis gel 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 (GraphPad, San Diego) for the estimation of kcat and Km values.

LC-MS/MS Analysis of Oligonucleotide Products from pol {eta} Reactions—pol {eta} reactions were performed for 6 h at 37 °C in 50 mM Tris-HCl (pH 7.8) buffer containing 1 mM dithiothreitol, 50 µg of bovine serum albumin ml–1, 50 mM NaCl, and 5 mM MgCl2 (200 µl total reaction mixture). The reactions were done with all four dNTPs at 1 mM each, 5 µM oligonucleotide substrate, and pol {eta} concentrations of 1.25–2.5 µM. The reactions were terminated by extracting the dNTPs using a spin column (Bio-Spin 6, Bio-Rad). The initial concentrations of Tris-HCl, EDTA, and dithiothreitol were restored in the above filtrate, and 15 units of E. coli UDG was added to the mixtures. The reactions were allowed to incubate at 37 °C for 4 h. Piperidine was added to the reaction mixtures (to 0.25 M), which were heated at 95 °C for 1 h followed by lyophilization. The residues were dissolved in a 70-µl volume for LC-MS/MS analysis.

For most of the experiments, MS analysis was performed in the Vanderbilt facility on a DecaXP ion trap instrument (ThermoFinnigan, San Jose, CA), using the general methods described earlier (37, 38). Samples were injected using an autosampler, with 10 µl withdrawn from the reaction mixture. The mixtures were separated on a Jupiter microbore column (1.0 mm x 150 mm, 5 µm; Phenomenex, Torrance, CA). Buffer A contained 10 mM NH4CH3CO2 (pH 6.8) and 2% CH3CN (v/v). Buffer B contained 10 mM NH4CH3CO2 (pH 6.8) and 98% CH3CN (v/v). The flow rate was 1.0 ml min–1, and the gradient was as follows: 0–2 min, hold at 100% A; 2–20 min, linear program to 100% B; 20–30 min, hold at 100% B; 30–32 min, linear program to 100% A; 32–40 min, hold at 100% A (for next injection). Oligonucleotides eluted at tR 9–12 min. Electrospray conditions (negative ion) were as follows: source voltage 3.4 kV, source current 8.5 µA, sheath gas flow rate setting 28.2, auxiliary sweep gas flow rate setting 4.3, capillary voltage 49 V, capillary temperature 230 °C, tube lens voltage 67 V. MS/MS settings were as follows: normalized collision energy 35%, activation Q 0.250, time 30 min, 1 scan. Product ion spectra were acquired over the range m/z 300–2000. The most abundant ions were selected for CID analysis. The calculations of the CID fragmentations of a certain oligonucleotide sequence were done using a program from the Mass Spectrometry Group of Medicinal Chemistry at the University of Utah.

In the case of the sequence analysis of pol {eta}-catalyzed full-length extension products across the C8-IQ adduct at the G1 position, the initial analysis was equivocal, and subsequent LC-MS/MS was performed on a Waters Acquit HPLC system (Waters, Milford, MA) connected to a Finnigan LTQ mass spectrometer (thermoelectric) using an Acuity HPLC BE C18 column (1.7 µm, 1.0 mm 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; 3–4.5 min, linear gradient to 80% A; 4.5–5 min, linear gradient 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 % A. The temperature of the column was maintained at 50 °C. Samples (10 µl) were infused with an autosampler. ES 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 345–2000. The triply charged species (m/z 1152.7) were used for CID analysis. The calculations of the CID fragmentations of oligonucleotide sequences were also done using a program linked to the Mass Spectrometry Group of Medicinal Chemistry at the University of Utah.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Extension of Primers Opposite C8- and N2-IQ Adducts in Templates—Initial studies were done with all four dNTPs using each of the human DNA polymerases available ({delta}, {eta}, {iota}, and {kappa}). Comparisons were made with the templates containing the C8- and N2-IQ adducts at the G1 and G3 positions, with the cognate primers (Figs. 2 and 3).

pol {delta}, in the presence of PCNA, readily extended the primers opposite the unmodified template but was totally blocked by either IQ adduct at both the G1 or G3 positions (Figs. 2 and 3). As expected, the translesion polymerases ({eta}, {iota}, and {kappa}) extended the primer opposite the unmodified template but with more less than full-length products due to their distributive character.


Figure 4
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  		FIGURE 4.
Approach to analysis of extension products by LC-MS/MS. The procedure involves extension of a primer beyond the G-IQ adduct in the template (C8-IQ adduct at G3 position in this example), digestion of the product with UDG and LC-MS/MS.

 
pol {eta} extended the primer opposite the C8-IQ adduct at the G1 or G3 position, although apparently better at G1 (Fig. 2). Extension also occurred opposite the N2-IQ adduct at both the G1 and G3 sites, although the products appeared to be less than full-length (Fig. 3). The latter phenomenon could be due to a lack of ability to read completely through or to deletions (see below).

Both pol {iota} and {kappa} showed very limited ability to catalyze primer extension opposite the templates with the C8- or the N2-IQ adduct (Figs. 2 and 3). Only with the N2-IQ adduct (both G1 and G3 sites) was any extension beyond a single residue seen (with pol {kappa}) (Fig. 3).


Figure 5
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  		FIGURE 5.
Analysis of the pol {eta} extension product of the primer extended opposite the template containing the C8-IQ adduct at the G3 site using LC-MS/MS. A, mass spectrum of the oligonucleotide peak. B, CID mass spectrum (of the m/z 1048.1 ion).

 
Steady-state Kinetics of 1-Base Incorporation into Primers Opposite C8- and N2-IQ Adducts—The steady-state kinetic parameters kcat, Km, and particularly kcat/Km provide a rough quantitative estimate of the ability of polymerases to insert bases opposite an adduct. A misincorporation factor, f (=(kcat/Km)wrong dNTP/(kcat/Km)dCTP), provides an index of the tendency to misincorporate for comparisons (39).

Preliminary experiments indicated that the dominant incorporations opposite both the C8- and N2-IQ adducts were polymerase-dependent as follows: C and A for pol {eta}, C and T for pol {iota}, and C and G for pol {kappa} (Tables 1 and 2). The activity with pol {delta} was too low to measure (Figs. 2 and 3). The efficiencies of insertion (kcat/Km) were 1–4 orders of magnitude lower than for insertion of dCTP opposite G, depending on the particular system. dCTP insertion was preferred to other dNTPs in all but two cases (f > 1), both with pol {iota} (Tables 2 and 3). Of the various insertions (C8- and N2-IQ adducts at G1 and G3 sites), the efficiency of pol {eta} insertion of dCTP was the most efficient for the adducts in all cases.


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  		TABLE 2
Steady-state kinetic parameters for 1-base incorporation opposite G1 by human pol {eta}, {iota}, and {kappa}

 


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  		TABLE 3
Steady-state kinetic parameters for one-base incorporation opposite G3 by human pols {eta}, {iota}, and {kappa}

 
LC-MS/MS Analysis of Bases Inserted during Primer Extension—LC-MS/MS analysis was used to identify the extension products from the pol {eta} reaction (with the other polymerases, the limited amounts of extended products were insufficient for any analysis). Mass spectra derived from ES ionization and CID are well suited for direct sequencing of oligonucleotides, and the method has the advantage of high sensitivity and application to modified oligonucleotides (37, 38). The same 19-mer primer used for full extension and single nucleotide insertion studies was redesigned to contain a single uracil residue (Fig. 4). Following polymerase extension of the primer positioned opposite the C8-IQ adduct at the G3 site in the template, the extended primers were digested by UDG followed by piperidine treatment to generate shorter oligonucleotides, which are more easily analyzed than the full-length primer product (Fig. 4). The CID spectra of longer oligonucleotide sequences are much more difficult to interpret when compared with shorter length sequences. After HPLC separation, an oligonucleotide-containing fraction was eluted at 10.55 min. Initial analysis of the ES mass spectrum indicated that m/z 1048.6 was the M – 3H ion (Fig. 5) and indicative of one major species present in the peak at tR 10.55 min. CID analysis of the m/z 1048.6 peak is shown in Fig. 5B. The composition of the m/z 1048.6 (M-3H) oligonucleotide was 1 phosphate, 5 Gs, 3 Cs, 1 T, and 1 A. Given the composition and sequence of the template, the unknown primer extension product was determined to be 5'-pGGCGCCGAGT-3', which is the full-length error-free bypass product (Fig. 6). The CID spectra matched well with those calculated based on the candidate sequence, with all of the major ions from Fig. 5 appearing in the calculated sequence (Table 4). The CID analysis was also compared with that of a standard oligonucleotide purchased from Midland Certified Reagent Co. (Midland, TX) (5'-pGGCGCCGAGT-3') (shown in supplemental Fig. S5).


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  		TABLE 4
Experimental versus theoretical fragmentation for product of pol {eta} extension experiment with C8-IQ adduct at the G3 site

 
A similar result was observed with the C8-IQ adduct at the G1 position; the error-free product accounted for 95% of the extended primer. However, one additional dATP was incorporated at the end of the completed primer strand, in a blunt end addition (Fig. 6). These results are presented in supplemental Fig. S6.4 An additional product (~5%) was also detected, but we have not been able to analyze the central section of the nucleotide sequence.


Figure 6
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  		FIGURE 6.
Proposed schemes for extension of primers. A, IQ adducts at the G3 position; B, IQ adducts at the G1 position.

 

LC-MS/MS analysis was also used to identify the extension products past the N2-IQ adducts of G at both positions of the NarI sequence (G3 and G1). Following pol {eta} extension and UDG digestion of the primer extended opposite the N2-IQ adduct at the G3 site of the template, an oligonucleotide-containing peak was eluted at tR 10.56 min during HPLC separation. Preliminary analysis of the ES mass spectrum indicated that m/z 1111.6 was the M – 2 ion (Fig. 7), indicating one major species present in the peak at 10.56 min. The possible composition of the m/z 1111.6 (M – 2H) oligonucleotide was 1 phosphate, 4 Gs, 2 Cs, and 1 A. Given the composition and sequence of the template, the unknown primer extension product was determined to be 5'-pGGCCGAG-3', a –2 deletion product (Fig. 6). CID analysis of the m/z 1111.6 peak is shown in Fig. 7. The CID spectra matched well with those calculated based on the candidate sequence, with all of the major ions from Fig. 7 appearing in the calculated sequence (Table 5). The CID analysis was also compared with that obtained for a standard commercial oligonucleotide (Midland) (5'-pGGCCGAG-3') and is shown in Fig. 8.


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  		TABLE 5
Experimental versus theoretical fragmentation for product of pol {eta} extension experiment with N2-IQ adduct at the G3 site

 
The same LC-MS/MS analysis was carried out for the oligonucleotide primer extended opposite the N2-IQ adduct at the G1 site (supplemental Fig. S7). The major extension product was a blocked one, 5'-pGGCGCC-3', in which the polymerase was unable to process to the end of the sequence. The LC-MS/MS results indicated that only C was inserted in the primer extension product that was analyzed (Fig. 6). However, steady-state kinetic analysis had indicated that pol {eta} inserted both dCTP and dATP opposite the G1 position N2-IQ G adduct, with nearly equal efficiency (Table 2). In light of this apparent discrepancy, we examined the further extension of a primer with A inserted opposite the G1 N2-IQ adduct and found no activity (supplemental Fig. S8). Thus, we conclude that both C and A can be inserted but that pol {eta} extends only beyond the N2-IQ G:C pair.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
One of the most interesting aspects of the heterocyclic amines is their extremely high mutagenicity (8, 9, 13). Most reports have focused on bacterial systems; the weaker mutagenicity seen in some mammalian cells has been attributed to the lack of the relevant bioactivation systems (40). In this work, we positioned both the N2- and C8-IQ adducts at both the G1 and G3 sites in the NarI sequence. In all cases, no replication of a primer occurred with human pol {delta}, even in the presence of PCNA, but the translesion polymerases pol {eta}, {iota}, and {kappa} could achieve some bypass. Of these, pol {eta} was clearly the most efficient (Tables 1 and 2). The steady-state kinetic analysis suggested that dCTP insertion was favored in all cases, indicative of fidelity. However, LC-MS/MS analysis of the pol {eta} primer extension products indicated that one of the four cases produced a frameshift mutation, with the N2-IQ adduct showing the common NarI CG/GC deletion only at the G3 position. Of the four cases, only the two C8-IQ adducts showed fidelity of replication. These in vitro results support the high inherent mutagenicity of IQ adducts and also show the major differences among individual chemistry of modifications, as well as with regard to the DNA site and individual polymerases.

Fuchs et al. (17) first reported that treatment of bacteria with N-acetoxy-AAF resulted in mutation at a hot spot, yielding a prominent –2 (GC) deletion in the center of a NarI site (GGCGCC). All three guanines in this sequence showed similar levels of adduction, but the G3 site was most "mutation-prone" (18). Subsequently, the same group was able to prepare plasmids in which each of the three guanines in the NarI sequence had been modified with (N-acetoxy) AAF (41), and only the G3 position yielded the –2 deletion (19). The Fuchs laboratory also demonstrated that IQ caused similar –1 and –2 deletions (42). The IQ-induced deletions were reported to be SOS-independent, in contrast to those seen with AAF, which seems surprising in light of the current knowledge about translesion synthesis (1, 4, 5). The –1 deletions induced by AAF have also been studied in runs of guanines and proposed to be the result of a slippage mechanism (43). Other subsequent work confirmed that the modification of G3 guanine of the NarI sequence with AAF produced an SOS-dependent frameshift in E. coli (23). The AF adduct at the same site produced only G to T transversions (in E. coli), and both AF and AAF produced G to T transversions in COS-7 kidney cells. Similar findings regarding G3 –2 deletions (in an E. coli system) for AAF G3 site adduct(s) were reported by Tebbs and Romano (29).


Figure 7
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  		FIGURE 7.
Analysis of the pol {eta} extension product of the primer extended opposite the template containing the N2-IQ adduct at the G3 site using LC-MS/MS. A, mass spectrum of the oligonucleotide peak. B, CID mass spectrum (of the m/z 1111.6 ion).

 
Attempts to understand the molecular basis of the –2 deletions have been made. Burnouf et al. (44) used AAF-modified oligonucleotides with several DNA polymerases (E. coli polymerase I (Klenow fragment, + or – exonuclease), bacteriophage T7 polymerase, E. coli pol III {alpha} subunit, and pol III holoenzyme). A variety of results was found, depending upon the polymerase. The G1 adduct (AAF) produced error-free replication, and the G2 and G3 sites yielded mutations. However, none of these polymerases produced the GC deletions. Gill and Romano (20), in contrast, reported that Klenow fragment (exonuclease–) produced a GC deletion when copying the NarI sequence with an AAF adduct at G3 but did not provide specific evidence. Crystal structures of two replicative DNA polymerases bound to AAF-modified DNA have been solved, with Bacillus stearothermophilus pol I (45) and bacteriophage pol T7 (46). AF-adducted G appears to pair with C; in both reports the C8-G AAF adduct induces an anti to syn shift in the glycosidic bond, which in the case of pol T7 leads to intercalation of the AAF fluorene ring into the hydrophobic pocket of the polymerase and locks it in the "open" conformation (46).

Less information is available to date about IQ adducts than AAF. As mentioned earlier, the guanine C8-IQ adduct is formed more readily in DNA than the N2-IQ adduct, but the N2-IQ accumulates in vivo due to slower repair (27, 28). IQ is mutagenic in human lymphoblastoid cells, but the limited mutation is probably the result of deficient bioactivation (40). The point should also be raised that there is a rather limited knowledge of which translesion DNA polymerases are present in various cell lines, which may be important (Fig. 1 and Tables 1 and 2). A –2 deletion (–GC) is a prominent mutation observed in transgenic (+lac) rats (liver, colon, and kidney) (30). Another heterocyclic amine adduct, the C8-guanyl derivative of 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine (commonly referred to as "PhIP"), has been examined in E. coli and COS-7 cells (47); G to T transversions and –1 deletions were observed, but the results were variable depending upon the sequence context.


Figure 8
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  		FIGURE 8.
MS of synthesized 5'-pGGCCGAG-3'. The synthetic oligonucleotide was used to confirm the assignment of the product identified in the primer extension opposite to the N 2-IQ adduct at the G3 template site (Fig. 7). A, mass spectrum; B, CID spectrum (of the m/z 1112.2 ion).

 
In our work presented here, the GC (2-base) deletion was restricted to the N2-IQ adduct at the G3 site of the NarI sequence (Fig. 4) and was only really seen with pol {eta}. The single-base insertion steady-state results (preferential insertion of dCTP) are misleading in that this is in all likelihood not a direct insertion opposite the modified G but rather opposite the G two bases 5' of the adduct. These findings with pol {eta} may provide some potential insight into the –2 deletion seen in vivo in rats (30).

pol {kappa} is able to bypass bulky lesions such as those formed from benzo[a]pyrene diol epoxide (48–51), although the kinetic parameters are difficult to compare in these cited studies because of the expression of units, and furthermore, sequence contexts can influence the outcomes (51). A role for pol {kappa} has been suggested for bypass of AAF C8-G adducts (52). However, in the context used, the conclusion was that (a truncated form of) pol {kappa} inserted dTTP opposite the AAF adduct (52). In our own work with the IQ adducts (Tables 2 and 3), pol {eta} was considerably more active than pol {kappa}. With some systems a two-step process has been invoked in which pol {eta}copies opposite lesions and then pol{kappa} proceeds to extend (48, 50, 53, 54). In the present work with the IQ adducts, pol {eta} could extend past the adducts without pol {kappa} (Figs. 2 and 3). Accordingly, we did not use mixtures of the polymerases in our work, which would have obfuscated quantitative measurements (Tables 2 and 3) and also the interpretation of LC-MS/MS analysis of products in the context of individual enzymes.

The NarI system has been used as a model for development of a system for studying the DNA site specificity of heterocyclic amine mutagenesis in vitro. However, some of the findings with the IQ adducts may not be directly relevant to the results obtained with AAF and vice versa. In addition, one concern about much of the earlier work with AAF is the identity of the adducts. Although the C8-guanyl adduct is the most plentiful in the treatment of DNA with N-hydroxy-IQ (27) or N-acetoxy-AAF (55, 56), some N2-guanyl adduct is formed, and oligonucleotides substituted with C8- and N2-guanyl adducts might not be readily discerned except with careful UV or MS CID analysis. The difference in the course of the primer extensions of the C8- and N2-IQ G3 adducts is remarkable (Fig. 4), and the possibility exists in a biological experiment (i.e. mutagenesis) that a trace of N2(–AAF) adduct might be responsible for activities attributed to the C8 adduct. For further understanding of IQ mutagenesis, one important need is analysis of x-ray structures of polymerases bound to IQ-modified oligonucleotides. Another need in this area is cellular analysis of which DNA polymerases are involved in translesion bypass and mutagenesis. Although the evidence presented here supports a role of pol {eta}, the analysis does not include all of the DNA polymerases potentially involved. Furthermore, the expression of the individual mammalian translesion DNA polymerases in various cell lines and tissues (in vivo) is not well characterized.

In conclusion, we have been able to show that IQ-dependent mutations are functions of the adduct chemistry, the DNA location, and the individual DNA polymerase involved. Although the literature already contains information for this general view with other types of DNA adducts, the point is clearly shown here with the altered adduct chemistry and with three of the major human translesion DNA polymerases. We have been able to dissect a mutagenic signature shown in mammals (a –2 base GC deletion) into components associated with different polymerases and sites. There are caveats about comparing human DNA polymerases with results in rats, but any comparisons with cells of human origin in culture have their own deficiencies. These results demonstrate a source of 2-base deletions provides a basis for further structural and biological experiments involving mechanisms and relevance.


   FOOTNOTES
 
* This work was supported in part by United States Public Health Service Grants R01 ES010375 (to F. P. G.), T32 ES007028 (to J. S. S.), and P30 ES000267 (to F. P. G. and C. J. R.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains Figs. S1–S8 and the characterization of the four modified template oligonucleotides (CGE and MS), MS of synthesized 5'-pGGCGCCGAGT-3' used to confirm the assignment of the product identified in the primer extension opposite the C8-IQ adduct at the G3 template site (Fig. 5 of text), analysis of the pol {eta} extension products of the primer extended opposite the template containing the C8-IQ and N 2-IQ adducts at the G1 site using LC-MS/MS, and analysis of the possible extension beyond a G1 site N 2-IQ G:A pair by pol {eta}. Back

1 Supported in part by a Merck fellowship. Back

2 To whom correspondence should be addressed: Dept. of Biochemistry and Center in Molecular Toxicology, Vanderbilt University School of Medicine, 638 Robinson Research Bldg., 23rd and Pierce Aves., Nashville, TN 37232-0146. Tel.: 615-322-2261; Fax: 615-322-3141; E-mail: f.guengerich{at}vanderbilt.edu.

3 The abbreviations used are: AAF, 2-acetylaminofluorene; AF, 2-aminofluorene; CGE, capillary gel electrophoresis; CID, collision-induced dissociation; ES, electrospray; IQ, 2-amino-3-methylimidazo[4,5-f]quinoline; LC, (high performance) liquid chromatography; MALDI-TOF, matrix-assisted laser desorption/time-of-flight; MS, mass spectrometry; PCNA, proliferating cell nuclear antigen; pol, DNA polymerase; UDG, uracil DNA glycosylase; HPLC, high pressure liquid chromatography. Back

4 This is the same major product that resulted from polymerization past the N 2-IQ adduct placed at the G3 site by Sulfolobus solfataricus DNA polymerase Dpo4 (J. S. Stover, H. Zang, G. Chowdhury, F. P. Guengerich, and C. J. Rizzo, submitted for publication). Back


   ACKNOWLEDGMENTS
 
We thank M. L. Manier for assistance with LC-MS/MS and K. Trisler for assistance in preparation of the manuscript.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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