Fig. 5.

POLD2 promotes inaccurate NHEJ. (A) Relative proportions of repair classes in CRITR CD4/CD71 translocation junctions in 293T cells transfected with nontargeting siRNA (siCTRL) or siPOLD2 #1. Exact, no sequence loss or gain; Deletion, only sequence losses; Insertion, only sequence gains; Complex, deletions coupled with insertions. (B) Absolute frequencies of modified and exact translocation junction sequences. Modified sequences include deletions, insertions, and complex. (C) Insertion lengths at CRITR CD4/CD71 translocation junctions in 293T cells. Insertion lengths include insertions found in complex junctions. (D) Schematic of deep-sequencing assay coupled with Hi-FIBR analysis of junctions at a single DSB. (E) DSB repair classes and (F) microhomology usage for intrachromosomal repair of CRISPR/Cas9-induced DSB in the CD4 locus in 293T cells transduced with siRNA-resistant POLD2 or control. Cells were transfected with the indicated siRNA followed by Cas9 and gRNA targeting CD4 48 h later and DNA was harvested 48 h after CRISPR transfection. (G and H) Repair classes and microhomology usage for intrachromosomal repair of CRISPR/Cas9-induced DSB in the ESR1 locus in 293T cells as in E and F. Data are presented as mean ± SE of n = 3 independent experiments. P values calculated using a two-way ANOVA with Tukey’s correction (A and E–H) or Student’s t test (C). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.