DNA Fiber Assay with PrimPol Variants

The stable cell line HeLa-shPrimPol has been described before^{24 (link)}. DNA sequences encoding WT PrimPol and Y100H variant were cloned into Gateway expression vectors (Invitrogen) introducing an N-terminal V5 tag. Transient transfection was performed using Lipofectamine 2000 (ThermoFisher, Waltham, MA, USA). HeLa-shPrimPol cells growing exponentially in culture were pulse-labelled with 50 μM CldU (20 min) followed by 250 μM IdU (20 min). Labelled cells were harvested and resuspended in phosphate buffered saline at 0.25 × 10⁶ cells/mL. Stretched DNA fibers were prepared as described^{55 (link)} with minor modifications. A detailed protocol is available upon request. For immunodetection of labelled tracks, fibers were incubated with primary antibodies anti-CldU (rat monoclonal anti-BrdU, Abcam #AB6326) and anti-IdU (mouse monoclonal anti-BrdU, BD Biosciences #347580) for 1 h at RT and the corresponding Alexa Fluor-conjugated secondary antibodies (Invitrogen/Molecular Probes #A-11007 and A-21121) for 30 min, both at room temperature in a humidity chamber. DNA was stained with anti-ssDNA (Millipore, #MAB3034) to assess fiber integrity. Fiber images were obtained in a DM6000 B Leica microscope. Fork rate was estimated from > 300 red-green tracks per condition using conversion factor 1 μm = 2.59 kb^{56 (link)}.