Kim et al. 10.1073/pnas.0505113102. |
Fig. 6. Schematic diagrams of assays used for measuring bypass efficiency and mutagenesis of thymine analogs
Fig. 7. Mutagenesis data for replication of thymine analogs in AlkB SOS bacteria, and data for frameshift mutagenesis.
Fig. 8. Representative thin-layer chromatogram showing data from mutagenesis experiments in Escherichia coli.
Supporting Materials and Methods
Nucleoside 5'-Triphosphate Synthesis (1, 2). The 0.1-mmol nucleoside was stirred in 0.25 ml of trimethylphosphate with proton sponge (32 mg, 0.15 mmol) at 0°C. POCl3 (10 ml) was added, and the mixture was stirred at 0°C. After 2 h, a mixture of 240 mg of tributyl ammonium pyrophosphate in 1 ml of anhydrous DMF and 0.1 ml of n-Bu3N was added to the reaction mixture under vigorous stirring at 0°C. After 5 min, 10 ml of 0.25 M aqueous triethylammonium bicarbonate buffer was poured into the solution. The aqueous layer was washed with dichloromethane (2 ´ 10 ml) and evaporated in vacuo.
The residue was purified by preparative TLC on a 20- ´ 20-cm silica gel (PK6F, 60 Å; layer thickness, 1,000 mm from Whatman; 2-propanol/concentrated NH3/water, 50:40:5). The second lowest band was excised and the elution solvent (10 ml) was added to the scraping. The scraping and solvent were homogenized on a vortex mixer and centrifuged, and the supernatant was decanted. After evaporation, the residue was purified by reverse-phase HPLC in a Zorbox ODS, 9.4-mm ´ 25-cm SB-C18 column with a 0-25 linear gradient of 40% CH3CN/0.1 M triethylammonium acetate, pH 7.5, for 30 min (for dHTP and dBTP). The eluent was evaporated under vacuum, and the triethylammonium acetate was removed by addition and evaporation of ethanol.
The residue of triethylammonium nucleoside triphosphate was dissolved in 0.4 ml of methanol and treated with a 1.2-ml acetone solution of 0.15 M sodium perchlorate. The precipitated sodium salt was collected by centrifugation, washed with acetone (4 ´ 1 ml), and dried under vacuum. 31P NMR spectra (160 MHz) were taken in deuterium oxide with 50 mM Tris, pH 7.5, and 2 mM EDTA. Trimethylphosphate was used as an external standard.
dHTP: 31P NMR 13.3 (d, J = 19.7 Hz), 14.2 (d, J = 19.9 Hz), 26.1 (t, J = 19.8 Hz); LR LC/MS (ESI-) mass calcd 447.04 for [C12H19O12P3-H], found 447.2.
dBTP: 31P NMR 11.9 (d, J = 18.3 Hz), 14.1 (d, J = 19.9 Hz), 25.6 (t, J = 19.9 Hz); LR LC/MS (ESI-) mass calcd 602.86 (51%), 604.85 (100%), and 606.85 (49%) for [C12H1779Br79BrO12P3-H], [C12H1779Br81BrO12P3-H], and [C12H1781Br81BrO12P3-H], found 602.9, 604.9, 606.9 in 1:2:1 isotope pattern.
Eckstein’s method (3) for dFTP, dLTP, and dITP
. General procedure for 5'-DMT, 3'-methoxyacetyl deoxyribosides. The specific 5'-DMT deoxyriboside (0.15 mmol) (4, 5) was coevaporated with dry pyridine twice and dissolved in 2 ml of pyridine. To the solution was added 40 ml of diisopropylethylamine (1.5 equiv), a catalytic amount of 4-(dimethylamino)pyridine, and 22 ml of methoxyacetyl chloride (1.6 equiv). The mixture was stirred at room temperature for 1 h, and then an additional portion of methoxyacetyl chloride (10 ml) was added to the solution. After another 1 h, the reaction mixture was quenched with 10 ml of methanol, and all volatiles were removed in vacuo. The residue was loaded onto a silica gel column (preequilibrated with 5% triethylamine in hexane) and eluted (40:10:1, hexane/ethylacetate/triethylamine).5'-DMT, 3'-methoxyacetyl dF: 1H NMR (CDCl3, ppm) 7.46 (d, 2H, J = 7.2 Hz), 7.37-7.32 (m, 5H), 7.27 (app t, 2H, J = 7.2 Hz), 7.202 (app t, 1H, J = 7.2 Hz), 6.82 (d, 4H, J = 8.8 Hz), 6.73(app t, 1H, JHF = 9.6 Hz), 5.44 (br d, 1H, J = 6.4 Hz), 5.30 (dd, 1H, J = 11.2, 5.2 Hz), 4.19- 4.16 (m, 1H), 4.06 (s, 2H), 3.77 (s, 6H), 3.45 (s, 3H), 3.43-3.34 (m, 3H), 2.42 (dd, 1H, J = 13.6, 4.8 Hz), 2.21-2.15 (m, 1H), 2.11 (s, 3H); 13C NMR (CDCl3, ppm) 170.0, 160.6 (dd, 1JCF = 239, 3JCF = 11 Hz), 158.7, 158.0 (dd, 1JCF = 238, 3JCF = 11 Hz), 145.0, 136.1, 130.3, 129.5 (app t, 3JCF = 6.8 Hz), 128.4, 128.3, 128.0, 127.0, 123.8 (dd, 2JCF = 12.9, 4JCF = 3.8 Hz), 120.9 (dd, 2JCF = 16.7, 3JCF = 3.8 Hz), 113.3, 103.2 (app t, 2JCF = 25 Hz), 86.6, 84.1, 77.8, 74.1, 70.0, 64.1, 59.6, 55.4, 40.7, 14.2; HRMS (MALDI+, CHCA matrix) calcd mass 641.2317 for [C36H36F2O7+Na], found 641.2306.
5'-DMT, 3'-methoxyacetyl dL: 1H NMR (CDCl3, ppm) 7.63 (s, 1H), 7.46 (d, 2H, J = 7.0 Hz), 7.36-7.33 (m, 4H), 7.29-7.21 (m, 4H), 6.82 (dd, 4H, J = 9.0, 1.5 Hz), 5.42 (d, 1H, J = 6.0 Hz), 5.36 (dd, 1H, J = 10.5, 5.5 Hz), 4.19 (dd, 1H, J = 5.5, 3.5 Hz), 4.07 (d, 2H, J = 3.5 Hz), 3.79 (s, 6H), 3.46-3.36 (m, 2H), 3.46 (s, 3H), 2.55 (dd, 1H, J = 13.5, 5.5 Hz), 2.21 (s, 3H), 2.06-2.00 (m, 13C NMR (CDCl3, ppm) 170.0, 158.7, 145.0, 137.8, 136.2, 136.0, 135.4, 133.9, 130.3, 129.4, 129.1, 128.1, 127.1, 113.4, 86.6, 84.2, 77.7, 70.0, 64.0, 59.7, 55.4, 40.4, 19.8; HRMS (FAB+, NBA matrix) calcd mass 673.1726 for [C36H36Cl2O7+Na], found 673.1769.
5'-DMT, 3'-methoxyacetyl dI: 1H NMR (CDCl3, ppm) 8.07 (s, 1H), 7.63 (s, 1H), 7.46 (d, 2H, J = 7.2 Hz), 7.37-7.35 (m, 4H), 7.30-7.18 (m, 3H), 6.83 (d, 4H, J = 8.8 Hz), 5.36 (d, 1H, J = 6.8 Hz), 5.10 (dd, 1H, J = 10.4, 4.8 Hz), 4.16 (dd, 1H, J = 6.0, 4.0 Hz), 4.05 (d, 2H, J = 3.2 Hz), 3.44 (s, 6H), 3.43-3.34 (m, 2H), 2.61-2.56 (m, 1H), 2.34 (s, 1H), 1.97-1.89 (m, 1H); 13C NMR (CDCl3, ppm) 169.9, 158.6, 144.9, 143.0, 142.8, 139.6, 137.0, 136.1, 136.0, 130.3, 128.3, 128.1, 127.0, 113.4, 101.7, 96.1, 86.6, 84.3, 83.0, 70.0, 63.8, 59.6, 55.4, 40.6, 27.4; HRMS (MALDI+, CHCA matrix) calcd mass 857.0443 for [C36H36I2O7+Na], found 857.0409.
General procedure for 3
'-methoxyacetyl deoxyribosides. The specific 5'-DMT, 3'-methoxyacetyl deoxyriboside (0.15 mmol) was dissolved into 80% aqueous acetic acid. The characteristic color of trityl cation was observed immediately. After 1 h, all volatiles were removed in vacuo, and the residue was purified on a silica gel column (60:1 to 30:1, chloroform/methanol).3'-Methoxyacetyl dF: 1H NMR (CDCl3, ppm) 7.26 (app t, 1H, JHF = 8.4 Hz), 7.20 (app t, 1H, JHF = 10.0 Hz), 5.36 (br d, 1H, J = 6.0 Hz), 5.22 (dd, 1H, J = 10.8, 5.2 Hz), 4.09 (s, 2H), 4.11-4.07 (m, 1H), 3.88-3.86 (m, 2H), 3.48 (s, 3H), 2.39 (ddt, 1H, J = 14.0, 5.2, 1.2 Hz), 2.15-2.07 (m, 1H); 13C NMR (CDCl3, ppm) 170.4, 160.0 (dd, 1JCF = 224, 3JCF = 11.3 Hz), 158.4 (dd, 1JCF = 223, 3JCF = 12.2 Hz), 129.5 9 (app t, 3JCF = 6.1 Hz), 122.7 (dd, 2JCF = 17, 4JCF = 3.8 Hz), 120.9 (dd, 2JCF = 17, 3JCF = 3.8 Hz), 103.6 (app t, 2JCF = 25.8 Hz), 85.4, 77.3, 74.9, 69.9, 63.1, 59.6, 40.0, 14.2; HRMS (DCI+, NH3) calcd mass 334.1460 for [C15H18F2O5+NH4], found 334.1467.
3'-Methoxyacetyl dL: 1H NMR (CDCl3, ppm) 7.41 (s, 1H), 7.35 (s, 1H), 5.34-5.29 (m, 2H), 4.11 (s, 2H), 3.94-3.90 (m, 2H), 3.48 (s, 3H), 2.58 (ddd, 1H, J = 14, 5.5, 1.5 Hz), 2.35 (s, 3H), 1.94-1.87 (m, 1H); 13C NMR (CDCl3, ppm) 170.5, 136.9, 135.5,134.1, 129.5, 128.5, 85.4, 77.0, 70.0, 63.3, 59.7, 39.9, 19.9; HRMS (DCI+, NH3) calcd mass 366.0869 for [C15H18Cl2O5], found 366.0881.
3'-Methoxyacetyl dI: 1H NMR (CDCl3, ppm) 7.83 (s, 1H), 7.63 (s, 1H), 5.30-5.28 (m, 1H), 5.07 (dd, 1H, J = 10.4, 4.8 Hz),4.09 (s, 2H), 3.94-3.88 (m, 2H), 3.46 (s, 3H), 2.61 (ddd, 1H, J = 13.6, 5.2, 1.2 Hz), 2.34 (s, 3H), 1.86-1.79 (m, 1H); 13C NMR (CDCl3, ppm) 170.4, 143.3, 142.1, 139.8, 136.6, 101.6, 96.3, 85.5, 82.8, 70.0, 63.2, 59.7, 40.1, 27.4; HRMS (DEI+) calcd mass 531.9243 for [C15H18I2O5], found 531.9249.
The 0.1-mmol methoxyacetyl nucleoside was coevaporated with 2 ml of dry pyridine, and the reaction flask was filled with argon. Anhydrous pyridine (100 ml) was injected through the septum followed by 300 ml of anhydrous dioxane. A freshly prepared 1 M solution of 2-chloro-4H-1,2,3-dioxaphosphorin-4-one in 110 ml of anhydrous dioxane was then injected into the well stirred solution of the nucleoside. After 10 min, a well vortexed mixture of a solution of 100 mg of tributylammonium pyrophosphate in 300 ml of anhydrous DMF, and 100 ml of tri-n-butylamine was quickly injected. The white precipitate immediately dissolved, and, 10 min later, a solution of 158-mmol iodine in 2 ml of 98%:2% (vol/vol) pyridine/water was then added. After 15 min, excess iodine was destroyed by adding a 5% aqueous solution of Na2S2O3, and the reaction solution was evaporated to dryness. The residue was dissolved in 10 ml of water. After standing at room temperature for 30 min, 20 ml of concentrated ammonia was added.
After 1 h, the solution was evaporated and the residue was purified by DEAE cellulose column (catalog no. D3764, Sigma) with a 0~100% linear gradient of water/1.0 M triethylammonium bicarbonate at pH 7.5 for 6 h. After evaporation, the residue was purified by reverse-phase HPLC in a Zorbox ODS, 9.4-mm ´ 25-cm SB-C18 column with a 0-50% linear gradient of CH3CN/50 mM triethylammoninum acetate (pH 7.5) for 20 min. The eluent was evaporated under vacuum, and the TEAA was removed by addition and evaporation of ethanol.
dFTP: 31P NMR 14.0 (d, J = 19.8 Hz), 14.3 (d, J = 19.9 Hz), 26.3 (t, J = 19.8 Hz); LR LC/MS (ESI-) mass calcd 483.02 for [C12H17F2O12P3-H], found 483.0.
dLTP: 31P NMR 13.5 (d, J = 19.1 Hz), 14.2 (d, J = 19.8 Hz), 25.9 (t, J = 19.0 Hz); LR LC/MS (ESI-) mass calcd 514.96 (100.0%), 516.95 (64.0%) for [C12H1735Cl2O12P3-H], [C12H1735Cl37ClO12P3-H], found 515.1 (100%), 517.1 (64%).
dITP: 31P NMR 13.5 (d, J = 18.9 Hz), 14.3 (d, J = 19.9 Hz), 26.2 (t, J = 19.9 Hz); LR LC/MS (ESI+) mass calcd 722.79 for [C12H17I2O12P3+Na], found 722.5.
Modified Oligonucleotide Synthesis.
Phosphoramidite derivatives of 3-toluene-1-b-D-deoxyriboside (dH), 2,4-dichloro-5-toluene--1-b-D-deoxyriboside (dL), 2,4-dibromo-5-toluene--1-b-D-deoxyriboside (dB), 2,4-diiodo-5-toluene--1-b-D-deoxyriboside (dI) were prepared as described in ref. 5. The phosphoramidite derivative of analog 2,4-difluoro-5-toluene--1-b-D-deoxyriboside (dF) was purchased from Glen Research (Sterling, VA). DNA oligonucleotides were synthesized on an Applied Biosystems 394 synthesizer using standard b-cyanoethylphosphoramidite chemistry. Oligomers were synthesized in DMT-off mode, deprotected in concentrated NH4OH at 55°C for 8 h, purified by preparative 20% denaturing polyacrylamide gel electrophoresis, isolated by the crush and soak method, and quantitated by absorbance at 260 nm.Molar extinction coefficients were calculated by the nearest neighbor method. Values for oligonucleotides containing nonnatural residues were obtained in the following way: The molar extinction coefficients at 260 nm for dH, dF, dL, dB, and dI were taken as 250, 1,000, 250, 500, and 3,900 M–1·cm–1, respectively. The individual extinction coefficients for all of the bases in a given oligomer were summed and compared with the sum from the corresponding sequence in which the nonnatural residues were replaced by T. Because in most cases the content of nonnatural residues in the sequences is low, this estimation method is unlikely to generate large errors in concentration.
Oligonucleotides containing one T or T analog for in vivo replication studies were characterized by MALDI-TOF mass spectrometry as described in ref. 5. Masses obtained for DNA 16-mer molecules are as follows: T, 4,881.15 observed (4,881.19 calculated); H, 4,847.21 observed (4,847.19 calculated); F, 4,883.24 observed (4,883.17 calculated); L, 4,916.04 observed (4,916.08 calculated); B, 5,004.95 observed (5,004.99 calculated); and I, 5,099.04 observed (5,098.99 calculated).
Steady-State Kinetics. 32P labeling of primer.
Primer (final concentration, ≈100 nM) was labeled with 5'-[g-32P]ATP (catalog no. PB10218, Amersham Biosciences, which is now GE Healthcare) and T4 polynucleotide kinase (catalog no. 18004-010, Invitrogen) and purified by using ethanol precipitation (6).Steady-state kinetics.
Steady-state kinetics for single-nucleotide insertions were carried out as described in refs. 7 and 8. Briefly, insertion reactions were carried out in 10-ml volumes in the reaction buffer: 50 mM Tris·HCl, pH 7.5/10 mM MgCl2/1 mM DTT/50 mg/ml BSA for exonuclease-free Klenow enzyme DNA polymerase I (catalog no. E70057Z, Amersham Biosciences).Labeled primer (final concentration, ≈20 nM), template, and unlabeled primer were mixed in a 2´ reaction buffer; each gave a final total concentration of primer–template of »20 mM. The primer–template duplexes were annealed by heating to 90°C and slow-cooling to 4°C over 1 h.
The commercial polymerase showed evidence of contaminating pyrophosphate in control reactions. By pretreating the polymerase enzyme with »0.0002 units of inorganic pyrophosphatase per unit of Klenow fragment (KF) for 10 min at room temperature before adding the primer– template duplex, interfering pyrophosphorolysis reactions were prevented.
A 2´ concentrated stock solution of duplex was mixed with 2´ KF exonuclease-deficient polymerase for 2 min at 37°C, and the reaction was initiated by adding a 2´ solution of the appropriate dNTP in buffer A (200 mM Tris·HCl/20 mM MgCl2·H2O/6 mM 2-mercaptoethanol, pH 8.0). Enzyme concentration and reaction time were adjusted in different dNTP reactions to give 1–20% incorporation in time periods £90 min. The following ranges of enzyme concentrations (units/ml) and times (min) were used (N®M denotes dNTP inserted across from base M in the template strand): H®A, 0.1, 16; F®A, 0.005, 2; L®A, 0.005, 0.33; B®A, 0.005, 2; I®A, 0.005, 6; T®A, 0.005, 0.33; H®G, 0.4, 30; F®G, 0.4, 40; L®G, 0.1, 18; B®G, 0.4, 20; I®G, 0.4, 90; T®G, 0.1, 3; H®C, 0.4, 38; F®C, 0.4, 20; L®C, 0.1, 16; B®C, 0.1, 38; I®C, 0.4, 90; T®C, 0.1, 18; H®T, 0.4, 20; F®T, 0.4, 13; L®T, 0.1, 1; B®T, 0.1, 1; I®A, 0.1, 7; T®A, 0.1, 50; A®H, 0.005, 18; G®H, 0.1, 70; C®H, 0.1, 30; T®H, 0.1, 20; A®F, 0.005, 0.83; G®F, 0.1, 60; C®F, 0.1, 15; T®F, 0.1, 5; A®L, 0.005, 0.5; G®L, 0.1, 30; C®L, 0.1, 40; T®L, 0.1, 5; A®B, 0.005, 1.5; G®B, 0.1, 60; C®B, 0.1, 25; T®B, 0.1, 5; A®I, 0.005, 14; G®I, 0.1, 60; C®I, 0.1, 30; T®I, 0.1, 10; A®T, 0.005, 0.5; G®T, 0.1, 3; C®T, 0.1, 20; T®T, 0.1, 30.
Reactions were quenched with 15 ml of loading buffer I [80% formamide/1´ TBE (89 mM Tris·borate/2 mM EDTA, pH 8.3)/0.05% xylene cylanol/bromophenol blue]. Extents of reaction were determined by running quenched reaction samples on a 20% denaturing polyacrylamide gel to separate unreacted primer from insertion products.
Steady-state kinetics analysis
. Radioactivity was quantified by using a PhosphorImager (Molecular Dynamics) and the IMAGEQUANT program. Relative velocity v was measured as the ratio of the extended product (Iext) to remaining primer (Iprim) as follows v = Iext/[(Iprim+ Iext)·t], where t represents the reaction time, and normalized for the lowest enzyme concentration used. The apparent K and V values were obtained from Hanes–Woolf plots.In Vivo
Bypass And Mutagenesis Assays. In vivo studies. The approach for studying the effect of differently sized T-isostere analogs on the efficiency and fidelity of replication is outlined in Fig. 6.Competitive replication of adduct bypass (CRAB) bypass efficiency analysis
. The data shown in Fig. 4 were generated using a modification of the CRAB assay (9), which allows both lesion bypass and mutagenesis analysis data to be obtained from a single transfection. Briefly, the concentrations of viable lesion-bearing and control (T at lesion site) genomes containing the sequence 5'-GAAGACCTXGGCGTCC-3' (X is T, H, F, L, B, I, or THF chemically stable abasic site) were equalized and mixed with ≈10% of a competitor M13 genome 3 bases longer containing the sequence 5'-GAAGACCTGGTAGCGCAGG-3', after which the mixture was electroporated into HK81 (AB1157, nalA) and HK82 (as HK81 but alkB22) Escherichia coli in triplicate, grown in liquid culture, and worked-up as described in ref. 9 but using a multiwell HT-200 electroporator (one pulse for five samples) set at 2,500 V, 125 ohms, 50 mF (Harvard Apparatus, Holliston, MA). If the lesion is a blockade to replication, there will be an increase in the percentage of output from the competitor, which is measured by PCR amplification of DNA from the progeny. The primer 5'- YCAGCTATGACCATGATTCAGTGGAAGAC-3' is identical in sequence to the vector and 5' end of the lesion-bearing or control oligonucleotide inserts, as well as the entire 28 bases in the competitor genome, allowing for specific amplification of these genomes; the primer 5'-YCAGGGTTTTCCCAGTCACGACGTTGTAA-3' anneals only to the invariant region of the M13 vector and does not span the insert region (Y is an aminoethoxyethyl ether group). PCR products were worked-up as described for the restriction endonuclease and postlabeling analysis of mutation frequency (REAP) assay (9), whereby the output mixture was cut with BbsI, 5' end-labeled at the newly exposed lesion site, trimmed with HaeIII, and subjected to 20% PAGE. Band intensity from the lesion output (18-mer) was divided by that of the competitor output (21-mer), and this ratio was divided by that obtained for the output from a nonlesion (T at lesion site) and competitor control mixture to obtain the percentage of lesion bypass in vivo. Each lesion was electroporated in triplicate for each cell type, and the average is reported with one standard deviation.REAP mutagenesis fidelity analysis
. The data shown in Fig. 5 were generated by using the REAP assay, which ultimately radiolabels the site that contained the lesion, and partitioning of the radioactivity on a TLC plate provides the base composition and, hence, the mutation frequency and specificity (9). The intensity of the 5'-32P-(lesion site)-18-mer output signal was substantially less in situations when the lesion was strongly blocking and the primers described above for the CRAB bypass assay were used; therefore, equally robust signals were achieved by specific amplification of progeny from the lesion-bearing genome using the primers 5'-YCAGCTATGACCATGATTCAGTGGAAGAC-3' and 5'- YTGTAAAACGACGGCCAGTGAATTGGACG-3', which are specific for both vector-insert junctions (increasing the annealing temperature from 64°C to 67°C eliminates the trace amount of competitor signal still present). After digestion with BbsI (to expose the site that had once contained the lesion), radiolabeling, HaeIII trimming, and PAGE purification of the [5'-32P]18 mer, the oligonucleotide was gel-purified and digested to 5'-[32P]dNMPs, which were partitioned on a TLC plate and quantified by PhosphorImagery as described (9). The average is reported with one standard deviation.1. Yoshikawa, M., Kato T. & Takenishi, T. (1967) Tetrahedron Lett. 8, 5065–5068.
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