Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Universitat de Barcelona, 08036, Barcelona, Spain.
Centre for Chromosome Stability (CCS), University of Copenhagen, 2200, Copenhagen, Denmark.
Cell Mol Life Sci. 2020 Feb;77(4):735-749. doi: 10.1007/s00018-019-03206-1. Epub 2019 Jul 11.
During S phase, replication forks can encounter several obstacles that lead to fork stalling, which if persistent might result in fork collapse. To avoid this collapse and to preserve the competence to restart, cells have developed mechanisms that maintain fork stability upon replication stress. In this study, we aimed to understand the mechanisms involved in fork stability maintenance in non-transformed human cells by performing an isolation of proteins on nascent DNA-mass spectrometry analysis in hTERT-RPE cells under different replication stress conditions. Our results show that acute hydroxyurea-induced replication blockade causes the accumulation of large amounts of single-stranded DNA at the fork. Remarkably, this results in the disengagement of replisome components from nascent DNA without compromising fork restart. Notably, Cdc45-MCM-GINS helicase maintains its integrity and replisome components remain associated with chromatin upon acute hydroxyurea treatment, whereas replisome stability is lost upon a sustained replication stress that compromises the competence to restart.
在 S 期，复制叉可能会遇到导致叉stall 的几个障碍，如果持续存在，可能会导致叉崩溃。为了避免这种崩溃并保持重新启动的能力，细胞已经开发了在复制应激下维持叉稳定性的机制。在这项研究中，我们旨在通过在不同复制应激条件下对 hTERT-RPE 细胞中的新生 DNA-质谱分析进行蛋白质分离，来了解非转化人细胞中叉稳定性维持的机制。我们的结果表明，急性羟基脲诱导的复制阻断导致大量单链 DNA 在叉处积累。值得注意的是，这导致复制体成分与新生 DNA 脱离，而不会损害叉的重新启动。值得注意的是，Cdc45-MCM-GINS 解旋酶保持其完整性，并且复制体成分在急性羟基脲处理后仍然与染色质相关联，而在持续的复制应激下，复制体稳定性丧失，从而损害重新启动的能力。
