T7 dna polymerase

A rolling-circle dsDNA template was prepared as previously described using T7 DNA polymerase (New England Biolabs) to extend a tailed oligonucleotide primer annealed to M13mp7(L2) ssDNA, synthesizing the complementary strand and generating a fork structure (41 (link)). Substrates were purified with phenol/chloroform extraction. Rolling-circle replication reactions with the E. coli replisome were performed as previously described (42 (link)), with: 30 nM DnaB (as hexamer), 40 nM Pol III core, 6.75 nM τ₃δδ’χψ, 30 nM β (as dimer), 600 nM DnaG and 500 nM SSB (as tetramer); 60 μM dNTPs supplemented with α-³²P-labeled-dATP, 250 μM UTP, GTP and CTP, and 1 mM ATP.
The Pol III replisome was loaded onto the fork structure by mixing Pol III core, β, clamp loader and helicase with dCTP, dGTP, ATP and 375 pM DNA substrate and incubating at 37°C for 5 min. Synthesis at 37°C was initiated by adding the dATP and dTTP, SSB and primase. Ten seconds after initiation, the indicated concentrations of Pol II or Pol IV were added. Reactions were quenched after 10 min by adding 25 mM EDTA and replication products were separated on a denaturing alkaline agarose gel (0.6%). The dried gel was exposed to a phosphor screen and imaged with a Personal Molecular Imager. The image displayed in Figure 8 is representative of two experiments.

The 5′-ends in mtDNA and nDNA from mouse liver were mapped using 5′-End-seq by treating 1 μg of total DNA with 0.3 M KCl for 2 h at 55 °C. Ribonucleotides were mapped by HydEn-seq (30 (link)) by hydrolyzing 1 μg of total DNA with 0.3 M KOH for 2 h at 55 °C. Afterward, ethanol-precipitated DNA fragments were treated for 3 min at 85 °C, phosphorylated with 10 U of phosphatase-free T4 polynucleotide kinase (New England BioLabs) for 30 min at 37 °C, heat-inactivated for 20 min at 65 °C, and purified with HighPrep PCR beads (MagBio). Phosphorylated products were treated for 3 min at 85 °C, ligated to oligo ARC140 (30 (link)) overnight at room temperature with 10 U of T4 RNA ligase, 25% polyethylene glycol (PEG) 8000, and 1 mM CoCl₃(NH₃)₆, and purified with HighPrep PCR beads (Mag-Bio). Ligated products were incubated for 3 min at 85 °C. The ARC76 ± ARC77 adaptor was annealed to the fragments for 5 min at room temperature. The second strand was synthesized with 4 U of T7 DNA polymerase (New England BioLabs) and purified with HighPrep PCR beads (MagBio). Libraries were PCR-amplified with KAPA HiFi Hotstart ReadyMix (KAPA Biosystems), purified, quantified with a Qubit fluorometric instrument (Thermo Fisher Scientific), and 75-base paired-end sequenced on an Illumina NextSeq500 instrument to locate the 5′-ends.