Synthetic oligodeoxyribonucleotides were from Oligos Etc, Inc. (Wilsonville, OR, USA) and The Midland Certified Reagent Co. (Midland, TX, USA). [α-32
P]dCTP and [α-32
P]ddATP (3000 Ci/mmol), and [γ-32
P]ATP (7000 Ci/mmol) were from GE HealthCare (Piscataway, NJ, USA) and Biomedicals (Irvine, CA, USA), respectively. Optikinase and terminal deoxynucleotidyl transferase were from USB Corp. (Cleveland, OH, USA) and Fermentas Inc. (Hanover, MD, USA), respectively. Protease inhibitor complete (EDTA-free) was from Roche Molecular Diagnostics (Pleasanton, CA, USA). Leupeptin, aprotinin and phenylmethylsulfonyl fluoride were from Calbiochem (La Jolla, CA, USA). Recombinant human Pol β was overexpressed and purified as described previously (16
). Human AP endonuclease (APE), uracil-DNA glycosylase (UDG) with 84 amino acids deleted from the amino terminus and DNA ligase I were purified as described previously (17–19
Overproduction and purification of the Pol θ polymerase domain
Based on the cDNA sequence of human Pol θ (1
), a 2.7-kb cDNA clone was amplified for overexpression and production of a 98-kDa C-terminal fragment of human Pol θ using a 5′-BamH1 primer: 5′-TGC CAA TCA TGA TGG ATC CTC ATC CCT CTT ACC-3′ and a 3′-Sal1 primer: 5′-CTC TGT TCT TTG CAG TCG ACT GCA TCT GCA C-3′. The amplified DNA was digested with BamH1 and Sal1 restriction enzymes, and the correct DNA fragment was gel-purified and ligated into BamH1–Sal1 digested pQE80L (Qiagen). The resulting plasmid pQE80L-θ2.7 was transformed into E. coli
BL21-CodonPlus-RP cells (Stratagene) for protein production.
Pol θ was purified from 1 to 2 l of E. coli BL21-CodonPlus-RP cells transformed with pQE80L-θ2.7 following induction with 1.0 mM IPTG at a temperature of 30°C for 3.5 h. After harvesting by centrifugation and storage at –80°C, cells were resuspended in buffer (35 ml) containing 50 mM Tris–HCl, pH 7.5, 150 mM NaCl, 0.5 mM 2-mercaptoethanol, 5% glycerol and a cocktail of proteinase inhibitors. Cells were disrupted in a French Press chilled to 4°C with a constant pressure of 25 000 psi. The lysate was clarified by centrifugation at 12 000 g for 20 min, and the supernatant was saved for further purification. Imidazole was added to the cleared lysate to a final concentration of 20 mM before being applied to a 2-ml Ni–NTA column (Qiagen). The resin was washed with 50 column volumes of buffer containing 500 mM NaCl, 1% Triton X-100, 50 mM Tris–HCl, pH 7.5 and 20 mM imidazole, followed by several column volumes of a second wash buffer containing 500 mM NaCl, 0.005% NP-40, 50 mM Tris–HCl, pH 7.5 and 20 mM imidazole. Bound proteins were step-eluted with the second wash buffer also containing 0.25 M imidazole. The buffer was exchanged on a Fast Desalt HR 10/10 column (GE Healthcare) equilibrated in 25 mM potassium phosphate, pH 7.5, 10% (v/v) glycerol, 1 mM EDTA, 1 mM 2-mercaptoethanol, 0.005% (v/v) NP-40 and 75 mM KCl. The desalted protein fraction was applied to a 1.0 ml HR 5/5 Mono S column equilibrated in 25 mM potassium phosphate, pH 7.5, 10% (v/v) glycerol, 1 mM EDTA, 1 mM 2-mercaptoethanol and 75 mM KCl. Protein was eluted with a linear KCl gradient (0.075–0.5 M) in this buffer, and fractions containing Pol θ were flash frozen in liquid nitrogen in small aliquots.
Generation of polymerase variant
The DNA polymerase activity of Pol θ was inactivated by disrupting the ability of two essential carboxylate side chains to bind Mg2+ within motif C of the polymerase active site. Polymerase domain residues Asp829 and Glu830, corresponding to residues 2540 and 2541 in full-length Pol θ, were changed to Asn and Gln, respectively, using the QuikChange Site-Directed Mutagenesis Kit (Stratagene) and the following oligonucleotides: forward Pol−: 5′-CAT CCT TCA ACT CCA TAA TCA ACT CCT ATA TGA AGT G-3′ and reverse Pol−: 5′-CAC TTC ATA TAG GAG TTG ATT ATG GAG TTG AAG GAT G-3′. The resulting plasmid bearing the polymerase activity mutations was sequenced in its entirety to confirm the D829N and E830Q substitutions. E. coli BL21-CodonPlus-RP cells were transformed with this plasmid, pQE80L-θPol−, followed by induction and purification of the protein as described above.
DNA polymerase assay
Pol θ activity was measured with a gel-based oligonucleotide extension assay as described previously (20–23
). An 18-mer primer (5′-TGA CCA TGT AAC AGA GAG-3′) was gel purified, labeled at the 5′ end with [γ-32
P]ATP and annealed in a 1:1.4 ratio to a 36-mer template (3′-ACT GGT ACA TTG TCT CTC GCA CTC ACT CTC TTC TCT-5′) in 10 mM Tris–HCl, pH 7.5 by heating to 90°C for 5 min followed by slow cooling to room temperature. One picomole primer/template (32
P-end labeled primer) was incubated with 5 nM Pol θ in a reaction mixture (10 μl) containing 25 mM HEPES, pH 7.5, 2 mM 2-mercaptoethanol, 0.1 mM EDTA, 5 mM MgCl2
, 50 µg/ml acetylated bovine serum albumin and 100 µM each dNTP, as indicated. Following incubation at 37°C for 10 min, reactions were stopped on ice by addition of 10 μl formamide gel-loading dye. Reaction products were separated by electrophoresis in a 15% polyacrylamide gel containing 8 M urea in 89 mM Tris–HCl, 89 mM boric acid, and 2 mM EDTA, pH 8.8. Imaging and data analysis were performed with a Typhoon PhosphorImager and the ImageQuant software (GE HealthCare).
Substrate preparation and dRP lyase activity assay
Preparation of the dRP lyase substrate and the dRP lyase assay conditions were essentially as described previously (24
). Briefly, the reaction mixture (20 µl) contained 50 mM HEPES, pH 7.5, 20 mM KCl, 2 mM DTT, 1 mM EDTA and 50 nM preincised 32
P-labeled AP site containing DNA. The reaction was initiated by adding the indicated amounts of Pol θ, mutant Pol θ or Pol β, followed by incubation at 37°C. Aliquots (9 µl each) were transferred at the indicated time intervals into the tubes that contained 1 µl of freshly prepared 200 nM NaBH4
. Reaction mixtures were shifted to 0–1°C (on ice) and incubation was continued for 30 min. After heating to 75°C for 2 min, the reaction products were separated by electrophoresis in a 15% polyacrylamide gel containing 8 M urea as described above. Imaging and data analysis were performed by PhosphorImager and ImageQuant software.
5′-End labeling and substrate preparation for dRP lyase and NaBH4 cross-linking reactions
Dephosphorylated 17-mer oligodeoxyribonucleotide (5′-UGTS-SGGATCCCCGGGTACBiotin-3′) containing a uracil residue at the 5′-end, a disulfide bond (S–S) 3 nt from the 5′-end, and biotin at the 3′-end was phosphorylated with Optikinase and [γ-32P]ATP. A 34-mer (5′-GTACCCGGGGATCCGTACGGCGCATCAGCTGCAG-3′) template was then annealed with 15-mer (5′-CTGCAGCTGATGCGC-3′) and 17-mer 32P-labeled oligonucleotides by heating the solution at 90°C for 3 min and allowing the solution to slowly cool to 25°C. The 32P-labeled duplex DNA was treated with human UDG to generate the 32P-labeled deoxyribose sugar phosphate containing single nucleotide gapped substrate. The S–S bond was included in the substrate molecule to enable future studies on cross-linking within the dRP lyase active site.
Covalent cross-linking of DNA to Pol θ
trapping technique was utilized to covalently cross-link Pol θ to DNA (25
). The reaction mixture (10 μl) contained 50 mM HEPES, pH 7.4, 20 mM KCl, 1 mM EDTA, 200 nM 32
P-labeled UDG-treated duplex DNA, 150 nM wild-type Pol θ, mutant Pol θ or 20 nM Pol β and 1 mM NaBH4
. Reactions were incubated first on ice for 60 min and then for 10 min at room temperature. After these incubations, 10 µl SDS–PAGE gel-loading dye was added to each reaction, boiled for 5 min and protein–DNA cross-linked complexes were separated by electrophoresis in a 10% NuPAGE Bis–Tris gel with a MOPS running buffer system. Radioactive bands were visualized with a Typhoon PhosphorImager.
Kinetic measurements of dRP lyase activity of Pol θ
Kinetic analysis of the dRP lyase activity of Pol θ was performed with a 32P-labeled 34-bp substrate that had been pretreated with UDG to generate a 32P-labeled dRP flap within a single-nucleotide gap in the duplex DNA. The reaction mixture contained 50 mM HEPES, pH 7.4, 20 mM KCl, 1 mM EDTA and 100 nM 32P-labeled UDG-treated duplex DNA. For the time course experiment, the reaction mixture (50 µl) was assembled at 0–1°C in the above buffer. Reactions were initiated by adding 400 nM Pol θ or the dilution buffer (control), as indicated in figure legends, and incubated at 37°C. Aliquots (9 µl each) were withdrawn at different time intervals and transferred to 0–1°C to stop the reaction. DNA products were stabilized by addition of 20 mM NaBH4 and incubated for 30 min on ice. Then, an equal volume of gel-loading buffer was added, and the reaction mixture was incubated at 75°C for 2 min. The reaction products were separated by electrophoresis in a 15% polyacrylamide TBE-Urea gel (Invitrogen, Pre-cast gel) for 30 min at constant voltage (200 V). To quantify the reaction products, gels were scanned on a PhosphorImager and the data were analyzed as above. Reaction rates were determined by plotting the amount of substrate released as a function of time, and data were fitted to the appropriate equation by nonlinear least squares methods. To examine the influence of protein concentrations on dRP removal, the reactions were assembled on ice, as above, and initiated by adding appropriate dilutions of Pol θ, as indicated in the figure legends. Reaction mixtures were incubated at 37°C for 10 min and processed as above.
In vitro SN-BER
Repair assay was performed in a final reaction volume of 30 μl. A 35-bp oligonucleotide duplex DNA (250 nM) containing uracil at position 15 was incubated in a BER reaction mixture that contained 50 mM HEPES, pH 7.5, 0.5 mM EDTA, 2 mM DTT, 20 mM KCl, 4 mM ATP, 5 mM phosphocreatine, 100 μg/ml phosphocreatine kinase, 0.5 mM NAD, 15 nM UDG, 15 nM APE, 200 nM DNA ligase I and 400 nM or 800 nM purified Pol θ, as indicated. Repair reactions were initiated by the addition of 10 mM MgCl2 and 2.2 μM [α-32P]dCTP (specific activity, 1 × 106 dpm/pmol), followed by incubation at 37°C. Aliquots (9 µl) were withdrawn at the indicated time intervals. Reactions were terminated by the addition of an equal volume (9 µl) of DNA gel-loading buffer. After incubation at 75°C for 2 min, the reaction products were separated by electrophoresis in a 15% polyacrylamide gel containing 8 M urea, and the data were analyzed as above.
Limited proteolysis and amino-terminal sequencing of Pol θ
The purified human Pol θ 98-kDa polymerase domain (66 µg) was subjected to limited proteolysis by mixing with trypsin (0.66 µg) at a 1:100 weight ratio (trypsin:Pol θ) in 100 mM Tris–HCl, pH 8.0 and then incubating the solution at 25°C. The final reaction mixture volume was 90 µl. Aliquots (20 µl each) were withdrawn at 5-, 15-, 30- and 60-min time intervals. A portion of each sample (18 µl) was mixed immediately with 10 µl SDS–PAGE gel-loading buffer, boiled for 5 min and separated by electrophoresis in a 12% NuPAGE Bis–Tris gel with a MOPS running buffer system. Proteins were electrophoretically transferred onto an Immun-Blot PVDF membrane (7 × 8.4 cm) (Bio-Rad) using a transfer buffer that contained 10 mM CAPS [3-(cyclohexylamino)-1-propanesulfonic acid] and 10% methanol, pH 11.0 at 50 V (7 V/cm) for 1 h. The membrane was stained briefly with 0.2% (w/v) Coomassie blue R-250 in 45% methanol and 10% acetic acid and destained with 90% methanol and 10% acetic acid. The membrane was air-dried, and protein bands were cut with a scalpel. Amino-terminal sequencing was performed using a Model 492 Procise sequencing system (Applied Biosystems) at Wake Forest University, Winston-Salem, NC. The remaining portion of the trypsin digested sample (2 µl) was subjected to NaBH4 cross-linking to a 5′-end labeled dRP lyase DNA substrate, as described above. The resulting gel was subjected to phosphorimaging, and then the same gel was silver-stained for protein detection.