Ultrapure ntps

Inorganic salts, acids, bases, and organic compounds were purchased from Merck (Kenilworth, NJ, USA), Sigma Aldrich (St Louis, MO, USA), and AppliChem (Darmstadt, Germany). Chromatography resins for protein purification used in this work were Ni-NTA Superflow purchased from Qiagen (CITY, Germany) and Heparin Sepharose Fast Flow to GE Healthcare (Fairfield, CT, USA). Ultrapure NTPs, dNTPs, and ddNTPs were supplied by GE healthcare (Fairfield, CT, USA). Labelled nucleotides and [α-³²P]dGTP were purchased from Perkin Elmer (Waltham, MA, USA). T4 polynucleotide kinase for DNA labelling was obtained from New England Biolabs (Ipswich, MA, USA) and used as indicated by the manufacturer. Vent polymerase was supplied by New England Biolabs (Ipswich, MA, USA). DNA oligonucleotides were synthesized by Sigma Aldrich (St Louis, MO, USA) and purified by 8 M urea-20% polyacrylamide gel electrophoresis. Oligonucleotides containing 8-oxoG were purchased from Eurogentec (Seraing, Belgium).

Pol I was purified, and in vitro experiments were performed as previously described (17 (link), 18 (link)). Briefly, the template sequence included the native promoter and rDNA sequence, except for a stretch of six Gs in the template strand. This enables synchronization of the polymerases at that position with the omission of CTP and is key for elongation rate assays. The sequences used in vitro were cloned from positions in the rDNA 5′ ETS (+405 to +604) and 18S (+2246 to +2445) and placed immediately downstream of the native rDNA promoter and the initially transcribed 55-nucleotide C-less stretch. The rDNA position is numbered with respect to the transcription start site. In vitro reactions (20 μl each) were performed in 1× transcription buffer (20 mM Tris–OAc [pH 7.9], 100 mM potassium glutamate, 8 mM magnesium acetate, 2 mM DTT, RNasin Plus [Promega], 0.2 mg/ml acetylated bovine serum albumin), 10 μCi of [α^32-P] UTP (PerkinElmer), 10 μM ultrapure NTPs (GE Life Sciences), and quenched with 1 ml of 1.25 M ammonium acetate in 95% ethanol.

Ultrapure NTPs and dNTPs were purchased from GE Healthcare and Roche, respectively. All oligonucleotides were synthesized by Lofstrand Laboratories (Gaithersburg, MD) and gel-purified prior to use.