All solvents, reagents and fine chemicals used in the molecularbiological and biochemical part are commercially available (Sigma‐Aldrich, Acros, Merck, Fluka, Roth, TCI, MCAT, Riedel de Haën or Amersham Pharmacia Biotech) and are qualified for molecular biologiy and biochemistry. Milli‐Q water was genereted with Easy pure UV/UF pure water system (Werner).
Small‐molecule analogues SM1, 4, 10, 12, 16, 17, 21, 23, 27‐32, and 35‐58 are of synthetic origin, whereat SM1, 4, 10, 16, 17, 21, 23, 27, and 28 were synthesized in my privious diploma thesis76 (see chapter 7). SM2, 3, 5‐9, 11, 13‐15, 18‐20, 22, and 24‐26 were purchased from the suppliers Maybridge, Labotest OHG and Key Organics. (‐)‐
Epigallocatechin gallate (EGCG), betulinic acid, oleanolic acid and lithocholic acid were purchased from Sigma Aldrich to evaluate the discovered small‐molecule inhibitors and the PEX assays. All compounds were stored in 10 or 5 µM DMSO stock solutions at ‐20°C.
8.2) Expression and purification of pol λ and pol β
Human recombinant pol λ and pol β were expressed and purified as described in literature55,243. Pol β‐wt ORF in pGDR11 plasmid and pol λ‐wt in pGDR11 plasmid in E.coli strain XL10 Gold was kindly provided by Bettina Bareth.75 The purity of the enzymes was
>95% as controlled by SDS‐PAGE analysis (Figure 36). Enzyme concentrations were determined by the Bradford assay244 using standart protocols (Roth).
Figure 36. SDS‐PAGE analysis of the recombinant human DNA polymerases. Lane M = marker (kDa), lane 1 =
8.2.1) Nucleotide‐ and amio acid sequences of pol β and pol λ
Coding DNA sequence of 6xHis‐tagged pol λ‐wt ORF in pGDR11:
8.3) SDS‐PAGE
Denaturing sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) was performed to separate proteins. SDS‐PAGE was performed using a stacking along with a separating gel. First, a 12% separating gel was prepared by mixing 1.7 mL H2O, 1.25 mL SDS‐
PAGE separating gel buffer, 50 μL 10% SDS, and 2 mL 30% bis‐acrylamide with 50 μL 10% APS and 5 μL TEMED. The solution was applied to the gel chamber and covered with 2‐
propanole. After polymerization, 2‐propanole was removed and the stacking gel including the gel pockets was prepared (1.22 mL H2O, 0.5 mL SDS‐PAGE stacking gel buffer, 10 μL 10%
SDS, 270 μL 30% bis‐acrylamide, 20 μL 10% APS, and 5 μL TEMED). Protein samples were mixed with one sixth of 6 SDS‐PAGE loading buffer and denaturated (5 min, 95°C).
PageRuler unstained protein ladder (Fermentas) was used as a standard. Separation was achieved by applying 35 mA in 1x SDS‐PAGE electrophoresis buffer. Afterwards the gel was stained according to standard protocols using coomassie brilliant blue staining‐ and destaining solutions (Carl Roth) respectively.
8.4) Nucleotides and oligonucleotides
dNTPs were purchased from Roche, [γ‐32P]ATP from Hartmann Analytics and oligonucleotides were purchased from Purimex and purified as described in chapter 8.4.1 Employed oligonucleotide sequences: Primer F20H, 5`‐d(CGT TGG TCC TGA AGG AGG AT);
template F33A, 5`‐d(AAA TCA ACC TAT CCT CCT TCA GGA CCA ACG TAC).
8.4.1) DNA oligonucletide purification
DNA oligonucletides were purified via PAGE. First, a 12% gel was prepared by reacting 120 mL 25% acrylamide‐bisacrylamide in 8.3 M urea, 105 mL 8.3 M urea, and 25 mL 8.3 M urea in 10 TBE buffer with 1.8 mL 10% APS in H2O and 90 μL TEMED. Final gel thickness was 1.5 mm. DNA samples containing denaturing PAGE loading buffer (stop solution) were separated by applying up to 100 W and 3000 V, respectively, in 1x TBE buffer at up to 45°C.
Afterwards DNA was visualized by UV light absorption (shadowing) and excised with a scalpel. Gel pieces were scrambled by pushing them trough a syringe. DNA was eluted by
adding H2O and incubating the mixture at 55°C overnight. The mixture was filtered using a syringe with a pad of silanised glass‐fibres wool. Finally, DNA was purified by ethanol precipitation. For DNA precipitation 0.1 volumes of 3 M NaOAc/HOAc (pH 5.3) followed by 2.5 volumes of 100% ethanol were added to the DNA sample. After incubation for >30 min at 20°C centrifugation was carried out at 4°C and 20,000×g for 30 min. The supernatant was discarded and 500 μL pre‐cooled 70% ethanol were added to wash the DNA pellet followed by another centrifugation step for 10 min. After removing the supernatant, the pellet was dried in vacuo (Speed‐Vac) and resolved in H2O. If the starting volume of the DNA sample was less than 1 mL volumes were adjusted.
To determine DNA concentrations, 1.5 to 2.0 μL sample were applied to the Nanodrop pedestals (Nanodrop ND1000). DNA was measured at 260 nm wavelength and the concentrations were calculated using the Lambert‐Beer law. The molar extinction coefficient for single stranded DNA resulted from the formula
ε[mM‐1cm‐1] = 15.2(A) + 12.01(G) + 8.4(T) + 7.05(C)
at which A, G, C and T are the numbers of the respective nucleotide in the sequence.
8.4.2) 5’‐Radioactive labelling of DNA oligonucletides
DNA oligonucleotide primers were radioactively labelled at the 5’ terminus by a 32P containing phosphate group using T4 polynucleotide kinase (PNK) (from Fermentas) which transfers the γ phosphate group from [γ‐32P]ATP (Hartmann Analytics) to the 5’ hydroxyl group. The reactions contained primer (0.4 μM), PNK forward reaction buffer (1), [γ‐32P]ATP (0.8 μCi μL‐1) and T4 PNK (0.4 U μL‐1) in a final volume of 50 μL and were incubated for 1 h at 37°C. The reaction was stopped by denaturing the T4 PNK for 2 min at 95°C and buffers and excess [γ‐32P]ATP were removed by gel filtration (MicroSpin Sephadex G‐25). Addition of unlabelled primer (20 μL, 10 μM) led to a final concentration of 3 μM of diluted radioactive labelled primer.
8.5) Radiometric primer extension
Enzyme activity, small molecule evaluation and IC50 measurements were done using a radiometric primer extension (PEX) assay with a radiometric product analysis. All primer
template complexes were annealed by heating a mixture of primer and template in the respective reaction buffer to 95°C for 5 min and subsequent cooling to room temperature prior to applications in all PEX assays.
8.5.1) Pol λ PEX assay with variable small‐molecule concentrations
Pol λ reactions were made in a final volume of 20 μL containing: 50 mM Tris‐HCl pH 7.5, 1.5 mM MgCl2, 5% (v/v) glycerol, 1 mM DTT (dithiothreitol), 250 nM purified recombinant pol λ, 150 nM radioactively labled primer F20H, 225 nM template F33A, 0.1 mg mL‐1 BSA (bovine serum albumin) (from Thermo Scientific), 1 μL compound solution (variable concentrations in DMSO) and accordingly 1 μL DMSO for the solvent control. Reactions and positive controls were started by addition of 5 μL dNTP solution (15 μM final). After 30 min incubation at 37°C, reactions and controls were subsequently quenched using 45 μL PAGE loading solution (formamide 80% (v/v), EDTA 20 mM, bromophenol blue 0.05% (w/v), xylene cyanol 0.05% (w/v)) per well. Then the reaction mixture was denaturated for 5 min at 95°C and analyzed by 12% PAGE containing 8 M urea. Visualization was performed using phosphorimaging.
8.5.2) Pol λ PEX assay with variable dNTP concentrations
Assay was carried out as described above (pol λ PEX assay with variable small‐molecule concentrations). Except that the inhibitor concentration was 50 μM and accordingly 0 μM for the solvent control. Positive control was started by addition of 5 μL dNTP solution (15 μM final). Reactions were started by addition of variable concentrations of 5 μL dNTP solution (15, 30, 60, 120, 240, 480 μM final).
8.5.3) Pol λ‐TdT assay with variable small‐molecule concentrations
Assay was carried out as described above (pol λ PEX assay with variable small‐molecule concentrations) with the exception that no template was used and the reaction was incubated for 120 min.
8.5.4) Pol β PEX assay with variable small‐molecule concentrations
Pol β reactions were made in a final volume of 20 μL containing: 50 mM Tris‐HCl (pH 7.9), 20 mM KCl, 5% (v/v) glycerol, 1 mM DTT, 2 mM MnCl2, 250 nM purified recombinant pol β, 150 nM radioactively labled primer F20H, 225 nM template F33A, 0.1 mg mL‐1 BSA, 1 μL compound solution (variable concentrations in DMSO) and accordingly 1 μL DMSO for the solvent control. Reactions and positive controls were started by addition of 5 μL dNTP solution (15 μM final). After 30 min incubation at 37°C, reactions and controls were subsequently quenched using 45 μL PAGE loading solution (formamide 80% (v/v), EDTA 20 mM, bromophenol blue 0.05% (w/v), xylene cyanol 0.05% (w/v)) per well. Then the reaction mixture was denaturated for 5 min at 95°C and analyzed by 12% PAGE containing
8 M urea. Visualization was performed using phosphorimaging.
8.6) Polyacrylamide gel electrophoresis (PAGE)
Denaturing polyacrylamide gels (12%) were prepared by polymerization of a solution of urea (8.3 M) and bisacrylamide/acrylamide (12%) in TBE buffer using ammonium peroxodisulfate (APS, 0.08%) and N,N,N’,N’‐tetramethylethylenediamine (TEMED, 0.04%). Immediately after addition of APS and TEMED the solution was filled in a sequencing gel chamber and left for polymerization for at least 45 min. After addition of TBE buffer (1) to the electrophoresis unit, gels were prewarmed by electrophoresis at 100 W for 30 min and samples were added and separated during electrophoresis (100 W) for approx. 1.5 h. The gel was transfered to whatman filter paper, dried (80°C, in vacuo, using a gel dryer model 583, BioRad) and exposed to a phosphor screen.
8.6.1) Quantitative analysis of gel images
After visualization of the gel, the image was analyzed with the software QuantityOne from BioRad. Every band of a lane was marked and quantified using the software. The generated percental values of every band of a lane were weighted with a factor (factor equates to the number of incorporated dNTPs) and summated. DMSO was used as solvent control and the activity of the enzyme in its presence was set to 100% conversion. Afterwards the
conversion of the reaction in presence of the compound (in their respective concentration) was calculated. The resulted conversions (in %) of independently conducted experiments were used to fit the data using the non‐linear regression calculation of Prism 4 (GraphPad).
Y=Bottom + (Top‐ Bottom) (1+10^((LogIC50‐X) HillSlope))‐1.