Journal: The Journal of Biological Chemistry
Article Title: Preferential Repair of Oxidized Base Damage in the Transcribed Genes of Mammalian Cells *
Figure Lengend Snippet: In vitro reconstitution of TC-BER. For radioactive incorporation into pUC-TS-5-OHU (with [α- 32 P]dTMP ( lanes 1–7 ) or [α- 32 P]dAMP ( lane 8 )), 10 pmol of substrate was incubated with 0.5 pmol of NEIL2 ( lanes 1 and 3–8 ), 25 fmol of PNK, 0.25 pmol each of Pol β and Lig IIIα, and an RNAP II fraction containing TBP, TFIIB, and E/F/H (Protein One, except in lane 5 without the transcription factors) in the presence (+) or absence (−) of NTPs, as described under “Experimental Procedures.” Lane 1 , undamaged DNA; lane 9 , end-labeled ϕX174 HinfI DNA marker (Promega). Complete repair is indicated by the [ 32 P]dTMP incorporation. The repaired plasmid was linearized with EcoRI/HindIII and analyzed in a 6% TBE-urea gel; the gel was dried and the reaction products were analyzed and quantitated with a PhosphorImager. Bottom , histograms indicating the -fold increase in repair activity; activity without NTPs ( lane 3 ) was set to 1. Error bars , S.E.
Article Snippet: The reactions were carried out at 30 °C for 90 min in a 50-μl reaction mixture and then stopped with 2% SDS in 0.2 m Tris-HCl (pH 7.5) and 10 μg/ml proteinase K. The nucleic acids were precipitated with ethanol, digested with EcoRI/HindIII, and once again ethanol-precipitated before final resuspension in formamide dye and denaturation at 90 °C for 3 min. Products were resolved alongside a φX174 HinfI DNA marker (Promega) on a 6% denaturing polyacrylamide gel in Tris borate-EDTA containing 8 m urea.
Techniques: In Vitro, Incubation, Labeling, Marker, Plasmid Preparation, Activity Assay