Ix71 fluorescence microscopy system

HeLa S3, XP12ROSV, and XP12ROSV/XPA cells were grown in DMEM supplemented with 15% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin, at 37°C in a humidified 5% CO₂ incubator. The cells were seeded into 24-well plates (Trueline), grown to about 90% confluence, and transfected with pBSII KS (-) FQ64 or pBSII KS (-) FQTT (20 μg), which was mixed with the transfection reporter24 (link) (10 pmol), using Lipofectamine 2000 (Life Technologies), according to the manufacturer's instructions. At the described intervals after transfection, the cells were analyzed with the Olympus IX71 fluorescence microscopy system. The fluorescence intensities of 10 cells in Fig. 4a were quantified and averaged, using the ImageJ software (National Institutes of Health).

Dual-incision assays (Supplementary Fig. S1d) were performed according to the described method35 (link), using the ³²P-labeled 140 bp duplexes with and without the (6–4) photoproduct (35 fmol) and the whole cell extracts36 (link) in 50 μl solutions for 2 h, followed by 14% denaturing PAGE and band detection with a Typhoon FLA 7000 image analyzer (GE Healthcare). Fluorescence intensities (Supplementary Fig. S1e) were measured at 37°C, using the same assay solutions after dilution to 200 μl with 1 M Tris-HCl (pH 8.0), on a JASCO FP-6500 spectrofluorometer equipped with an EHC 573 temperature controller. The excitation and emission wavelengths were 494 and 520 nm, respectively, with bandwidths of 3 nm. For the transfection of the XP12ROSV cells25 (link) (Supplementary Fig. S1f), the cells were grown in Dulbecco's modified Eagle's medium (DMEM) supplemented with 15% fetal bovine serum, 100 units/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified 5% CO₂ incubator. The cells were seeded into a 4-well slide lumox (Greiner Bio-One), grown to about 90% confluence, and transfected with the 140 bp duplexes using Lipofectamine 2000 (Life Technologies), according to the manufacturer's instructions. At 6 h after transfection, the cells were analyzed with an Olympus IX71 fluorescence microscopy system.