The alternative non-homologous end-joining (NHEJ) pathway promotes DNA double-strand break (DSB) repair in cells deficient for NHEJ or homologous recombination, suggesting that it operates at all stages of the cell cycle. Here, we use an approach in which DNA breaks can be induced in G1 cells and their repair tracked, enabling us to show that joining of DSBs is not functional in G1-arrested XRCC4-deficient cells. Cell cycle entry into S-G2/M restores DSB repair by Pol θ-dependent and PARP1-independent alternative NHEJ with repair products bearing kilo-base long DNA end resection, micro-homologies and chromosome translocations. We identify a synthetic lethal interaction between XRCC4 and Pol θ under conditions of G1 DSBs, associated with accumulation of unresolved DNA ends in S-G2/M. Collectively, our results support the conclusion that the repair of G1 DSBs progressing to S-G2/M by alternative NHEJ drives genomic instability and represent an attractive target for future DNA repair-based cancer therapies.

替代性的非同源末端连接（NHEJ）途径在缺乏NHEJ或同源重组的细胞中促进DNA双链断裂（DSB）修复，表明它在细胞周期的所有阶段均起作用。在这里，我们使用一种可以在G1细胞中诱导DNA断裂并对其修复进行跟踪的方法，从而使我们能够证明DSB的连接在G1阻滞的XRCC4缺陷细胞中不起作用。进入S-G2 / M的细胞周期可通过Pol依赖和PARP1独立的替代NHEJ恢复DSB修复，修复产物带有千碱基长的DNA末端切除，微同源性和染色体易位。我们确定了G1 DSBs下XRCC4和Polθ之间的合成致死相互作用，与未解决的DNA末端在S-G2 / M中的积累有关。总的来说，