Figure 5: Impact of loss of Pol θ and Lig4 on chromosomal DSB repair.

(a) Cellular sensitivity to etoposide. A clonogenic survival assay was performed with different concentrations of etoposide, a topoisomerase II inhibitor that induces chromosomal DSBs (mean±s.d.; n=3). Inset graph: Enlargement at low concentrations of etoposide. (b) Scheme for a Cas9-induced DSB joining assay. After DSB induction at the HPRT locus (exon 3), 6-thioguanine-resistant colonies (that is, HPRT-negative cells) were subjected to junction analysis. (c) Features and distribution of junctions obtained from wild-type, LIG4^−/− and LIG4-complemented LIG4^−/−POLQ^−/− (#3) cells. The features were classified into four categories; microhomology (2–6 bp homology), templated insertion (≥6 bp direct or inverted repeats), undefined insertion (1–12 bp) and 0–1 bp homology. A Fisher’s exact test was used to determine statistical significance. (*P<0.00001). Although not indicated, the absence of templated insertion in POLQ^−/− cells is statistically significant (P=0.0052). (d) Schematic representation of recombinants obtained from wild-type, LIG4^−/−, LIG4^−/−POLQ^−/−, LIG4-complemented LIG4^−/−POLQ^−/− (#3) and POLQ^−/− cells. (e) Schematic representation of four recombinants obtained from LIG4^−/−POLQ^−/− cells. The locations of Alu elements at the HPRT locus are also indicated. (f) Junction sequences in the four recombinants obtained from LIG4^−/−POLQ^−/− cells.