叉保护复合物中的 TIMELESS (TIM) 充当复制体的支架，以防止其解偶联并确保有效的 DNA 复制叉进展。然而，其协调前导链和滞后链合成以限制单链 DNA (ssDNA) 暴露的根本基础仍然难以捉摸。在这里，我们证明，在正在进行的 DNA 复制叉处 TIM 的急剧降解会诱导由缺陷的冈崎片段 (OF) 加工引起的 ssDNA 间隙的积累。缺乏 TIM 的细胞无法支持支持 LIG1 和 FEN1 介导的经典 OF 加工机制所必需的聚（ADP-核糖基）化。因此，XRCC1（一种已知的 PARP1 依赖性单链断裂修复效应子）向复制叉后面的复制后 ssDNA 间隙的募集受到损害。 TIM-PARP1 复合物的物理破坏反映了 TIM 的快速损失，表明 TIM-PARP1 相互作用对于激活该补偿途径至关重要。因此，FEN1 和 TIM-PARP1 相互作用的联合缺陷会导致协同 DNA 损伤和细胞毒性。我们认为 TIM 对于 PARP1 与复制体的结合以协调滞后链合成与复制叉进展至关重要。我们的研究将 TIM 确定为 OF 加工酶的合成致死靶点，可用于癌症治疗。© 作者 2024。由牛津大学出版社代表 Nucleic Acids Research 出版。

TIMELESS (TIM) in the fork protection complex acts as a scaffold of the replisome to prevent its uncoupling and ensure efficient DNA replication fork progression. Nevertheless, its underlying basis for coordinating leading and lagging strand synthesis to limit single-stranded DNA (ssDNA) exposure remains elusive. Here, we demonstrate that acute degradation of TIM at ongoing DNA replication forks induces the accumulation of ssDNA gaps stemming from defective Okazaki fragment (OF) processing. Cells devoid of TIM fail to support the poly(ADP-ribosyl)ation necessary for backing up the canonical OF processing mechanism mediated by LIG1 and FEN1. Consequently, recruitment of XRCC1, a known effector of PARP1-dependent single-strand break repair, to post-replicative ssDNA gaps behind replication forks is impaired. Physical disruption of the TIM-PARP1 complex phenocopies the rapid loss of TIM, indicating that the TIM-PARP1 interaction is critical for the activation of this compensatory pathway. Accordingly, combined deficiency of FEN1 and the TIM-PARP1 interaction leads to synergistic DNA damage and cytotoxicity. We propose that TIM is essential for the engagement of PARP1 to the replisome to coordinate lagging strand synthesis with replication fork progression. Our study identifies TIM as a synthetic lethal target of OF processing enzymes that can be exploited for cancer therapy.© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.