PARP1 ufmylation modulates replication stress response

高保真的DNA复制确保了父母的遗传信息准确地传递到子代细胞。研究细胞对复制应激反应的分子机制对衰老、组织动态平衡以及包括癌症和神经变性在内的发病机制具有重要意义。UFM1是最近发现的泛素样蛋白，通过E1样酶(UBA5)、E2型酶(UFc1)和E3连接酶(UFl1)与靶蛋白(S)偶联，这一过程称为ufm化。然而，超甲基化的生物学功能在很大程度上仍不清楚。我们发现，UFM1的缺陷通过延迟S阶段检查点激酶CHK1的激活来损害DNA复制应激反应。此外，还发现PARP1发生了超甲基化，从而增强了其酶活性。此外，缺陷的ufmylating会影响MRN复合体向DNA损伤部位的募集。我们先前证明了PARP1产物PAR与CHK1结合，以促进其在S阶段检查点的激活。因此，我们假设PARP1超甲基化促进CHK1介导的复制应激，并协调MRN介导的停滞叉子的修复。本项目旨在研究(1)超甲基化是否在S期检查点中发挥重要作用；(2)其潜在的分子机制；(3)其在体内的生物学功能。为了实现这些目标，我在德国弗里茨·利普曼研究所的实验室和深圳大学的徐兴志教授决定联合起来进行生化和细胞实验以及基因研究。具体地说，我们将研究ATR-CHK1途径是否直接受到ufm化的调控，以及PARP1 ufm化如何通过修复停滞不前的复制叉来协调对复制应激的反应。为此，我们将使用人类细胞系和小鼠模型，在这些模型中，ufmylating是转基因的。我们将进行一项生化研究，以定位PARP1中的ufm化位点，并测试其对CHK1激活和分叉稳定性的影响。最后，我们将通过产生和使用低PAR化或酶促死亡的PARP1细胞和小鼠来检查PAR化和ufm化的可能的相互作用。我们的研究将揭示UFM1信号在处理DNA复制压力和维持基因组稳定性方面的新功能。探索ATR-CHK1和PARP1/CHK1通路网络中ufm化的具体功能可能为将这一新的翻译后修饰过程调控为治疗人类恶性肿瘤和衰老相关疾病的新策略提供科学知识。

High-fidelity DNA replication ensures the accurate transmitting of parental genetic information to daughter cells. Studying the molecular mechanisms of the cellular response to replication stress has important implications on aging, tissue homeostasis, and pathogenesis, including cancer and neurodegeneration. UFM1 is the most recently identified ubiquitin-like protein and conjugates to target protein(s), a process known as ufmylation, via an E1-like enzyme (UBA5), an E2-like enzyme (UFC1), and an E3 ligase (UFL1). However, the biological function of ufmylation remains largely unknown. We found that defects in UFM1 compromise the DNA replication stress response by delaying the activation of the S-phase checkpoint kinase CHK1. Moreover, PARP1 is found ufmylated, which enhances its enzymatic activity. Furthermore, a defective ufmylation compromises the recruitment of the MRN complex to DNA damage sites. We showed previously that the PARP1 product PAR binds to CHK1 to facilitate its activation in the S-phase checkpoint. Thus, we hypothesize that a PARP1 ufmylation promotes CHK1-mediated replication stress and coordinates a MRN-mediated repair of the stalled forks. This project aims to study (1) whether ufmylation plays an important role in the S-phase checkpoint; (2) its underlying molecular mechanism, and (3) its biological function in vivo. To achieve these goals, my lab at the Fritz Lipmann Institute (FLI), Germany and Prof. Dr. Xingzhi Xu from the Shenzhen University, China decided to combine our effort to carry out biochemical and cellular experiments, as well as genetic studies. Specifically, we will investigate whether the ATR-CHK1 pathway is directly modulated by ufmylation and how PARP1 ufmylation coordinates the response to replication stress with the repair of stalled replication forks. To this end, we will employ human cell lines and mouse models, in which ufmylation is genetically modified. We will perform a biochemical study to map the ufmylation site in PARP1 and test its impact on the CHK1 activation and fork stability. Finally, we will examine a possible interplay of PARylation and ufmylation by generating and using hypoPARylated or enzymatic dead PARP1 cells and mice. Our research will reveal a novel function of UFM1 signaling in handling DNA replication stress and the maintenance of genome stability. Exploring the specific function of ufmylation in the network of the ATR-CHK1 and PARP1/CHK1 pathways may provide scientific knowledge to modulate this novel post-translational modification process as new therapeutic strategies for the treatment of human malignancy and aging-related diseases.