Escherichia coli dna polymerase 1

Detection of incorporated ribonucleotides was performed as previously described (Hiller et al., 2012 (link)). Briefly, 200 ng of human primary fibroblasts or yeast genomic DNA (wild-type and RNASEH2-deficient) was treated with 20 nM of purified recombinant human RNase H2 (Loomis et al., 2014 (link)) or water in RNase H2 reaction buffer (50 mM Tris-HCl pH 8, 60 mM KCl, 10 mM MgCl₂, 0.01% BSA (Bovine Serum Albumin), 0.01% Triton) at 37°C for 1 hr. 20 μM of unlabeled dATP, dGTP and dTTP, plus 3.7 × 10⁵ Bq [α-³²P]-dCTP (PerkinElmer, Santa Clara, CA) and 5 U of Escherichia coli DNA polymerase I (New England Biolabs, Inc., Ipswich, MA) were added and the reaction was incubated at 16°C for 30 min and was run on a 1% TAE (Tris-acetate-EDTA) agarose gel. Visualization was performed using a Storm PhosphorImager, and bands were quantified using ImageQuant (GE Healthcare, United Kingdom). Relative ribonucleotide loads were calculated by dividing the radiolabel incorporation in the RNase H2-treated sample over the untreated sample. Experiments were performed at least in triplicate.

First strand DNA was synthesized using SuperScript III Reverse transcriptase (Invitrogen, Carlsbad, USA) starting with 10 μg of RNA. For second strand DNA synthesis we used 30 units of Escherichia coli DNA Polymerase I (New England Biolabs, Ipswich, USA) in the presence of 2.5 units of RNAse H (Epicentre, Madison, USA), 5 units E. coli DNA Ligase (New England Biolabs, Ipswich, USA), Ligase buffer, DNA Polymerase I Buffer (NEB2) and 300 μM of dNTPs. Water was added to 100 μl and the mixture was incubated for 150 min at 16 °C. Subsequently 5 U of T4 DNA Polymerase was added and incubated for a further 30 min at 16 °C. The final products were cleaned using the cycle pure DNA clean up kit and stored at −20 °C.

Cells were fixed and permeabilized in CSK buffer supplemented with 1% PFA and 2.5% Triton X-100 for 15 min at room temperature. The cells were incubated overnight at 37 °C with a 70 μL nick translation reaction buffer containing 50 mM Tris-HCI (pH 7.5), 10 mM MgCl2, 1 mM DTT, 1 U of Escherichia coli DNA polymerase I (New England Biolabs, Ipswich, MA, USA), 10 μM each of dATP, dGTP, dCTP, and dTTP (Sileks, Moscow, Russia), and 3 μM fluorescein-labeled dUTP. The reaction was terminated by incubation the slides in PBS; the slides were then used for immunostaining. For positive control, fixed cells were treated with RNase-free DNase I (1 U/mL; New England Biolabs) for 30 min at room temperature in PBS.