Fig 6. POLQ modestly affects EJ repair and HDR, whereas BRCA2 promotes HDR to a much greater degree than RMR events.

(A) Influence of POLQ and RAD52 on EJ repair events induced by sgRNA/Cas9-mediated DSBs targeted to the 5' edge and 3' edge of the non-homologous insert (5' & 3' edge) in the EJ7ins reporter (S5 Fig). In this reporter, restoration of GFP expression occurs via EJ without indels. Shown are the percentages of GFP+ cells normalized to both transfection efficiency and to parallel EV samples (EV = 1). The RAD52^KO, POLQ^e16m, and RAD52^KOPOLQ^e16m EJ7ins reporter cells were transfected with expression vectors for the sgRNAs and Cas9, along with empty vector (EV), POLQ expression vector, or RAD52 expression vector, and parallel reactions that contained an oligonucleotide that had 14 nt of homology to the 5' and 3' GFP sequences (14-0-14) were tested for possible roles of oligonucleotide bridging of DSB ends. Two independent clones were tested for each cell line with two independent replicates for a total n = 4, and error bars represent SD. Experiments with siRNA were performed as in Fig 3. Two independent clones were tested for each reporter with two independent replicates for a total n = 4. Error bars represent SD. † P < 0.05 (unadjusted P-value), * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001, EV vs. complementation, using unpaired t-test with Holm-Sidak correction. (B) Influence of RAD52 and POLQ on the HDR reporter (DR-GFP). RAD52^KO, POLQ^e16m, and RAD52^KOPOLQ^e16m cells with stable transfection of DR-GFP were transfected with an sgRNA and Cas9 expression plasmid targeting DR-GFP, along with EV, RAD52 expression vector, or POLQ expression vector. Experiments with siRNA were performed as in (A). Error bars represent SD and n = 8. † P < 0.05 (unadjusted P-value), ** P < 0.01, **** P < 0.001, EV vs. complementation, and siCTRL vs. siRNA treatment using unpaired t-test with Holm-Sidak correction. (C) Immunoblot analysis shows the depletion of BRCA2 with siRNA in the parental line using siCTRL or a pool of four BRCA2 siRNAs (siBRCA2). Actin was a loading control. (D) Influence of BRCA2 depletion on chromosomal break repair events. Parental cells with each reporter were co-transfected with the sgRNA and Cas9 expression plasmid as indicated, along with siCTRL or siBRCA2. For the Δ7 reporter, oligonucleotides were co-transfected as indicated. GFP+ frequencies are normalized to transfection efficiency and shown relative to the non-targeting siRNA (siCTRL = 1). n = 4 for 5' & 3' edge, n = 6 for 5' edge and mid-ins, and n = 8 for Δ7 reporter. Error bars represent SD. † P < 0.05 (unadjusted P-value), * P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 0.001, siCTRL vs. siBRCA2, using unpaired t-test with Holm-Sidak correction.