Klenow polymerase

Fluorescence-based polymerase activity test was executed in 20 mM Tris-HCl pH 7 and 5 mM MgCl₂, with 3 μM of dT₂₄ overhang DNA template, 1 μM of FAM 5′-labelled DNA primer and various concentrations of either dATP or dZTP. The Klenow polymerase was added to final concentration of 5 U in 50 μl. The assay was conducted at 37 °C for 5 min. Before adding the protein, DNA was hybridized by heating up to 95 °C and gradually cooling to reaction temperature. Reactions were terminated by adding two volumes of a buffer containing 10 mM EDTA, 98% formamide, 0.1% xylene cyanol and 0.1% bromophenol blue, and stored at 4 °C. Products were preheated at 95 °C for 10 min, before being separated with polyacrylamide gel electrophoresis and visualized by FAM fluorescence on Typhoon FLA 9000 imager. All oligonucleotides were from Eurogentec, chemicals from Sigma-Aldrich, Klenow polymerase from Takara Bio, dATP from Fermentas (Thermo Fisher Scientific) and dZTP from TriLink BioTechnologies. Oligonucleotide sequences are specified in Supplementary Table 6.

Fluorescence polymerase activity tests were executed in 20 mM Tris–HCl pH 7 and 50 mM MgCl₂, with 3 μM of dT₂₄ overhang DNA template, 1 μM of FAM 5′-labeled DNA primer (Supplementary Table S2), varying concentration of dATP or dZTP and 0.83 μM (0.06 mg ml^–1) of DpoZ constructs (5 min of incubation at 37°C in 100 μl volume). The Klenow polymerase used as a control was at 5 U in 50 μl (10 min incubation) and was purchased from Takara Bio.
Before adding the protein, DNA was hybridized by heating up to 95°C and gradually cooling to reaction temperature. Reactions were terminated by adding two volumes of a buffer containing 10 mM EDTA, 98% formamide, 0.1% xylene cyanol and 0.1% bromophenol blue, and stored at 4°C. Products were preheated at 95°C for 10 min, before being separated with polyacrylamide gel electrophoresis and visualised by FAM fluorescence or radioactivity on Typhoon FLA 9000 imager. All oligonucleotides were ordered from Eurogentec, chemicals from Sigma-Aldrich, dATP from Fermentas (Thermo Fisher Scientific) and dZTP from TriLink BioTechnologies.
Intensities of the nucleic acid bands corresponding to fully extended primers were quantified with ImageJ (20 (link)); mid-points of dNTP concentrations needed for product saturation were determined by fitting a sigmoidal function to the data.