Department of Cancer Biology, Penn Center for Genome Integrity, Basser Center for BRCA, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.
Department of Biochemistry and Structural Biology and Greehey Children's Cancer Research Institute, University of Texas Health Science Center at San Antonio, San Antonio, TX, USA.
Nature. 2023 Jul;619(7968):201-208. doi: 10.1038/s41586-023-06177-3. Epub 2023 Jun 14.
Break-induced telomere synthesis (BITS) is a RAD51-independent form of break-induced replication that contributes to alternative lengthening of telomeres. This homology-directed repair mechanism utilizes a minimal replisome comprising proliferating cell nuclear antigen (PCNA) and DNA polymerase-δ to execute conservative DNA repair synthesis over many kilobases. How this long-tract homologous recombination repair synthesis responds to complex secondary DNA structures that elicit replication stress remains unclear. Moreover, whether the break-induced replisome orchestrates additional DNA repair events to ensure processivity is also unclear. Here we combine synchronous double-strand break induction with proteomics of isolated chromatin segments (PICh) to capture the telomeric DNA damage response proteome during BITS. This approach revealed a replication stress-dominated response, highlighted by repair synthesis-driven DNA damage tolerance signalling through RAD18-dependent PCNA ubiquitination. Furthermore, the SNM1A nuclease was identified as the major effector of ubiquitinated PCNA-dependent DNA damage tolerance. SNM1A recognizes the ubiquitin-modified break-induced replisome at damaged telomeres, and this directs its nuclease activity to promote resection. These findings show that break-induced replication orchestrates resection-dependent lesion bypass, with SNM1A nuclease activity serving as a critical effector of ubiquitinated PCNA-directed recombination in mammalian cells.
断裂诱导端粒合成（BITS）是一种 RAD51 非依赖的断裂诱导复制形式，有助于端粒的替代性延长。这种同源定向修复机制利用最小的复制体，包括增殖细胞核抗原（PCNA）和 DNA 聚合酶-δ，在多个千碱基上执行保守的 DNA 修复合成。这种长片段同源重组修复合成如何应对引发复制应激的复杂二级 DNA 结构尚不清楚。此外，断裂诱导复制体是否协调其他 DNA 修复事件以确保其连续性也不清楚。在这里，我们将同步双链断裂诱导与分离染色质片段的蛋白质组学（PICh）相结合，以在 BITS 期间捕获端粒 DNA 损伤反应蛋白质组。这种方法揭示了一个以复制应激为主导的反应，突出了通过 RAD18 依赖性 PCNA 泛素化驱动的修复合成驱动的 DNA 损伤容忍信号。此外，SNM1A 核酸酶被鉴定为泛素化 PCNA 依赖性 DNA 损伤容忍的主要效应因子。SNM1A 在受损端粒上识别泛素化的断裂诱导复制体，这指导其核酸酶活性以促进切除。这些发现表明，断裂诱导复制体协调依赖于切除的损伤绕过，SNM1A 核酸酶活性作为哺乳动物细胞中泛素化 PCNA 指导的重组的关键效应因子。
