The stabilization of stalled forks has emerged as a crucial mechanism driving resistance to poly(ADP-ribose) polymerase (PARP) inhibitors in BRCA1/2-deficient tumors. Here, we identify UFL1, a UFM1-specific E3 ligase, as a pivotal regulator of fork stability and the response to PARP inhibitors in BRCA1/2-deficient cells. On replication stress, UFL1 localizes to stalled forks and catalyzes the UFMylation of PTIP, a component of the MLL3/4 methyltransferase complex, specifically at lysine 148. This modification facilitates the assembly of the PTIP–MLL3/4 complex, resulting in the enrichment of H3K4me1 and H3K4me3 at stalled forks and subsequent recruitment of the MRE11 nuclease. Consequently, loss of UFL1, disruption of PTIP UFMylation or overexpression of the UFM1 protease UFSP2 protects nascent DNA strands from extensive degradation and confers resistance to PARP inhibitors in BRCA1/2-deficient cells. These findings provide mechanistic insights into the processes underlying fork instability in BRCA1/2-deficient cells and offer potential therapeutic avenues for the treatment of BRCA1/2-deficient tumors.

失速叉的稳定已成为驱动 BRCA1/2 缺陷肿瘤对聚（ADP-核糖）聚合酶（PARP）抑制剂产生耐药性的关键机制。在这里，我们确定 UFL1（一种 UFM1 特异性 E3 连接酶）是 BRCA1/2 缺陷细胞中叉稳定性和对 PARP 抑制剂反应的关键调节因子。在复制压力下，UFL1 定位于停滞叉并催化 PTIP（MLL3/4 甲基转移酶复合物的一个组成部分，特别是在赖氨酸 148 处）的 UFMylation。这种修饰促进了 PTIP-MLL3/4 复合物的组装，从而富集H3K4me1 和 H3K4me3 处于停滞状态，随后招募 MRE11 核酸酶。因此，UFL1 的缺失、PTIP UFMylation 的破坏或 UFM1 蛋白酶 UFSP2 的过度表达可以保护新生 DNA 链免遭广泛降解，并赋予 BRCA1/2 缺陷细胞对 PARP 抑制剂的抗性。这些发现为 BRCA1/2 缺陷细胞中叉不稳定的过程提供了机制见解，并为 BRCA1/2 缺陷肿瘤的治疗提供了潜在的治疗途径。