Fig. 4. Pol θ promotes alt-NHEJ of unrepaired G1-DSBs and translocations in S-G2/M phase.

a, b Representative semi-quantitative nested PCR analysis of Igk V_10-95-to-J₄ coding joins from untreated (NT), G1-blocked (ABLki) and released (Re) v-Abl pro-B cells of the indicated genotype. Il-2 gene PCR was used as a loading control. Released cells were harvested 5 days after washing off ABLki. Two independent cell lines were used for each genotype: Xrcc4^−/− p53^−/− (Xr15307-3 & Xr15307-11); Xrcc4^−/− Parp1^−/− p53^−/− (Xr-P1P2Fp53-3 & Xr-P1P2Fp53-4); Xrcc4^−/− Polq^−/− p53^−/− (Polq-2-307-Xr25 & Xr307-3-Polq35). Source data are provided as a Source Data file. The PCR gels are representative of n = 2 independent experiments. c Schematic representation of the Igk locus, with positions of the BACs used for generation of DNA FISH probes indicated. d Representative metaphases from released Xrcc4^−/− p53^−/−, Xrcc4^−/− Polq^−/− p53^−/− and Xrcc4^−/− Parp1^−/− p53^−/− v-Abl pro-B cells with Igk C BAC probe (red), Igk V BAC probe (green) and chromosome 6 paint (white). Yellow and orange arrowheads point to broken and translocated chromosome 6, respectively. Scale bars = 3 μm. The number of analyzed metaphases for each condition is indicated in Supplementary Table 6. e Percentage of aberrant metaphases from released v-Abl pro-B cells of the indicated genotypes. Released cells were harvested 5 days after washing off ABLki. Histograms represent means ± s.d. from at least two independent experiments performed with a minimum of two independent B cell clones (the list of independent experiments and total metaphases analyzed are provided in Supplementary Table 6). The mean percentage of total aberrant metaphases is indicated above. The mean percentages of metaphases with translocations (red) and chromosomes breaks (white) are indicated (See also Supplementary Table 6). ***P < 0.001 (Two-sided Fisher exact test).