Fig 2. UV exposure of cultured cells induces mutations preferably at the RPL13A TTCCG hotspot site independently of repair.

(a) Cultured human cells, either A375 melanoma cells or fibroblasts with NER deficiencies, were subjected to a single UVB dose (20 J/m²) during approx. 2 seconds. Following recovery, cellular DNA was subsequently assayed for subclonal mutation in the RPL13A -116 bp TTCCG promoter hotspot site (see Fig 1a and 1b, top row) using SiMSen-Seq error-corrected amplicon sequencing [23]. Non-UV-treated sample were included as controls. (b) 10 samples were sequenced at 311k to 950k reads each, resulting in 7.3k to 13.8k error-corrected reads at ≥10x oversampling. (c) Subclonal mutations in a 46 bp amplicon window encompassing the RPL13A -116 bp hotspot in A375 melanoma cells. The hotspot site and TTCCG element are indicated in gray/red, respectively. Positive axis, UV-treated sample; negative axis, no UV control. (d-g) As panel c but showing results from XPC -/- (lacking global NER), XPA -/- (lacking global and transcription-coupled NER), ERCC8 -/- and ERCC6 -/- (lacking transcription-coupled NER) mutant fibroblasts.