Fig. 6.
Elevated TMEJ repair signatures in cells and cancers with PolqSL gene mutations. a WT, 53bp1−/−, and Brca2Mut/− MEFs were transfected with Cas9 ribonucleoprotein (Cas9-RNP) targeting the Rosa26 locus. Forty-eight hours later the targeted region was amplified and analyzed by high throughput sequencing (Illumina MiSeq). b Percentage of repair products classified as NHEJ (≤5 bp del, or 1–3 bp insertion), TMEJ (>5 bp deletion and >2 bp MH), and “Other” (>5 bp deletion and 0–2 bp MH), for the indicated MEF genotypes. c The relative frequency (normalized to WT) of end joining products with >2 bp MH and deletion size within the indicated ranges. 53bp1−/− MEFs are associated with larger-sized TMEJ signature deletions. b, c Mean values ± SEM (n = 6) are shown. Significance determined using an unpaired, two-tailed t-test (*p < 0.05; **p < 0.01; ***p < 0.001). d, e DNA repair products are detected by digital PCR using WT MEFs transduced with the indicated PolqSL gene-targeting sgRNA. HR is detected after co-transfection of a homology donor. Relative rates (normalized to WT + sgCtrl) of (d) homologous recombination (HR) and (e) TMEJ (95 bp deletion with 5 bp MH) are indicated as mean values ± SEM (n = 3). Significance determined using an unpaired, two-tailed t-test (***p < 0.001; ****p < 0.0001). f–j Analysis of breast cancers in TCGA. PolqSL mutant breast cancers are identified using cBioPortal as having a truncating mutation or deep copy number deletion. POLQ mRNA expression (f) and COSMIC Signature 3 (g) are elevated in cancers with PolqSL gene alterations. h–j Whole exome sequencing (WES) and whole genome sequencing (WGS) analyses in TCGA breast cancers to detect MH-flanked deletions (MHD). h Breast cancers with PolqSL gene alterations are more likely to have a detectable MHD by WES than breast cancers without alteration in PolqSL genes. The error bar is a bootstrapped 95% confidence interval. WGS data is available for 94 TCGA breast cancers. i High correlation between MHD detected by WGS or WES. j Significantly higher MHD count, detected by WGS, among breast cancers with PolqSL gene alterations, relative to the non-altered breast cancers. f–g, i–j Statistical significance was assessed by two-tailed Mann–Whitney tests. *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001