Fig. 2: SSTR deletions of 2 bp are increased in RNase-H2-null HeLa cells.
From: Signatures of TOP1 transcription-associated mutagenesis in cancer and germline
a, Schematic of the reporter targeting the AAVS1 safe harbour locus to generate reporter cells (Extended Data Fig. 3). HA, homology arm; L, left; R, right. b, c, Validation of RNASEH2A-KO reporter clones. b, Immunoblot analysis of cell lysates detecting the three RNase H2 subunits. GAPDH was used as the loading control. Gel source data are provided in Supplementary Fig. 1. c, Cellular RNase H2 enzyme activity. Data are mean ± s.d. n = 3 technical replicates. HeLa, no modification; parental, HeLa with reporter (grey); KO1 and KO2, CRISPR-mediated RNASEH2A-KO clones (red); RNASEH2A+, CRISPR-edited reporter clone retaining RNase H2 activity (green). d, Fluctuation assays establish a significantly increased mutation rate in RNase-H2-null (KO) cells (P = 2 × 10−6). Statistical analysis was performed using a two-sided Mann–Whitney test. Data are median ± 95% confidence intervals. The data points show the rates for independent cultures. n = 9 (RNase H2 proficient, RNASEH2A+); n = 10 (KO1, open circles) and n = 6 (KO2, open squares). e, 2 bp SSTR and SNMH deletions are frequent in both RNASEH2A+ and RNASEH2A-KO cells. Indel mutation spectra. n shows the number of indels identified by sequencing colonies from independent cultures.
