Methyl 3h dttp

The primer extension ability of TkoPolD was measured by counting incorporated radioactivity into DNA strands using dNTP containing [methyl-³H]-dTTP as substrates, and the activities were compared among the WT, ΔPIP, ΔKR, and ΔPIPΔKR in the presence and absence of PCNA. The reaction was performed in 25 μl containing 20 mM Tris–HCl, pH 8.0, 100 mM NaCl, 10 mM KCl, 10 mM (NH₄)₂SO₄, 2 mM MgCl₂, 0.1% Triton X-100 and 0.1 mg/mL BSA, 10 nM template primer substrate (prepared by annealing M13mp18ssDNA and a deoxyoligonucleotide, M13-63; 5′-dTGCCAAGCTTGCATGCCTGCAGGTCGACTCTAGAGGATCCCCGGGTACCGAGCTCGAATTCGT-3′), 0.2 mM dNTPs including 0.13 μM [methyl-³H]-dTTP (PerkinElmer, MA), 20 nM PCNA, and 5 nM PolD, at 72 °C for 1, 2, and 4 min. The reaction mixture was pre-incubated for 3 min, and PolD was added to initiate the reaction. After incubation, aliquots (8 μl) were fractionated and spotted onto DE81 filters (GE Healthcare). The filters were washed with 5% Na₂HPO₄ solution thrice and dried. Incorporated radioactivity was measured with a scintillation counter Tri-Carb 3110TR (PerkinElmer).