Homologous recombination (HR) is an important DNA double-strand break (DSB) repair pathway that copies sequence information lost at the break site from an undamaged homologous template. This involves the formation of a recombination structure that is processed to restore the original sequence but also harbors the potential for crossover (CO) formation between the participating molecules. Synthesis-dependent strand annealing (SDSA) is an HR subpathway that prevents CO formation and is thought to predominate in mammalian cells. The chromatin remodeler ATRX promotes an alternative HR subpathway that has the potential to form COs. Here, we show that ATRX-dependent HR outcompetes RECQ5-dependent SDSA for the repair of most two-ended DSBs in human cells and leads to the frequent formation of COs, assessed by measuring sister chromatid exchanges (SCEs). We provide evidence that subpathway choice is dependent on interaction of both ATRX and RECQ5 with proliferating cell nuclear antigen. We also show that the subpathway usage varies among different cancer cell lines and compare it to untransformed cells. We further observe HR intermediates arising as ionizing radiation (IR)-induced ultra-fine bridges only in cells expressing ATRX and lacking MUS81 and GEN1. Consistently, damage-induced MUS81 recruitment is only observed in ATRX-expressing cells. Cells lacking BLM show similar MUS81 recruitment and IR-induced SCE formation as control cells. Collectively, these results suggest that the ATRX pathway involves the formation of HR intermediates whose processing is entirely dependent on MUS81 and GEN1 and independent of BLM. We propose that the predominant ATRX-dependent HR subpathway forms joint molecules distinct from classical Holliday junctions.

同源重组 (HR) 是一种重要的 DNA 双链断裂 (DSB) 修复途径，它从未损坏的同源模板复制在断裂位点丢失的序列信息。这涉及重组结构的形成，该结构经过处理以恢复原始序列，但也具有在参与分子之间形成交叉 (CO) 的潜力。合成依赖性链退火 (SDSA) 是一种 HR 亚途径，可防止 CO 形成，并被认为在哺乳动物细胞中占主导地位。染色质重塑剂 ATRX 促进了另一种可能形成 CO 的 HR 亚途径。在这里，我们表明 ATRX 依赖性 HR 在修复人体细胞中大多数两端 DSB 方面胜过 RECQ5 依赖性 SDSA，并导致 CO 的频繁形成，通过测量姐妹染色单体交换 (SCE) 进行评估。我们提供的证据表明，亚通路选择取决于 ATRX 和 RECQ5 与增殖细胞核抗原的相互作用。我们还表明，不同癌细胞系的亚通路使用情况不同，并将其与未转化的细胞进行比较。我们进一步观察到仅在表达 ATRX 且缺乏 MUS81 和 GEN1 的细胞中作为电离辐射 (IR) 诱导的超细桥出现的 HR 中间体。一致地，仅在表达 ATRX 的细胞中观察到损伤诱导的 MUS81 募集。缺乏 BLM 的细胞显示出与对照细胞相似的 MUS81 募集和 IR 诱导的 SCE 形成。总的来说，这些结果表明 ATRX 途径涉及 HR 中间体的形成，其加工完全依赖于 MUS81 和 GEN1 而独立于 BLM。