Studies on the mechanism of pathway choice during repair of ionizing radiation induced DNA double strand breaks

电离辐射诱导的DNA双链断裂修复途径选择机制研究

• 批准号: 415585014
• 负责人: Professor Dr. George Iliakis
• 金额: --
• 依托单位: Institut für Medizinische Strahlenbiologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2019
• 资助国家: 德国
• 起止时间: 2018-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/415585014?language=en
• 关键词: Studies; mechanism; pathway; choice; during

Ionizing radiation (IR) generates adverse effects, such as cell killing or carcinogenesis, by inducing DNA double strand breaks (DSBs). DSBs are severe lesions eliciting in cells the "DNA damage response (DDR)": a network of signal transduction pathways that extensively modify cell metabolism and set the stage for DSB processing, cellular adaptation, programmed cell death or senescence. DSBs are processed efficiently in cells. Yet, DSBs are linked to severe biological effects because their processing is associated with mutations and chromosomal translocations. The present project investigates how the DSB processing apparatus underpins these effects. Four pathways, operating on distinct principles, process DSBs: Homologous recombination repair (HRR), canonical non-homologous end joining (c-NHEJ), alternative end joining (alt-EJ) and single strand annealing (SSA). Surprisingly, only HRR is able to faithfully restore the genome while all remaining pathways, including c-NHEJ that is enzymatically dominant, tolerate/catalyze mutations and translocations. Thus, the choice of pathway determines whether genomic alterations accompany DSB processing. Here, we formulate and test a novel hypothesis regarding DSB repair pathway choice promising to advance our understanding regarding logic and necessities inherent to DSB processing. The central question is: How cells choose between c-NHEJ and HRR? We provide evidence that Ku translocation away from the DNA end and mediated by DNA-PK autophosphorylation in the initial steps of c-NHEJ, changes Ku protein conformation and induces an interaction with Mre11 that triggers resection and HRR. We show that under certain conditions well over 50% of DSBs may be processed by HRR making such a mechanism highly relevant to genomic integrity. A large set of promising preliminary experiments guide the development of three work packages testing interactions between Ku and Mre11 in vitro and in vivo, and analyzing their connections to the c-NHEJ- and HRR-apparatus, as well as to checkpoint responses. The proposed molecular characterization of the DSB processing apparatus will advance our understanding of genomic instability sources and will accelerate the development of strategies to improve radiation protection and radiation therapy.

电离辐射(IR)通过诱导DNA双链断裂(DSB)产生负面效应，如细胞死亡或致癌。DSB是一种严重的损伤，在细胞中引发“DNA损伤反应(DDR)”：一种广泛改变细胞新陈代谢的信号转导网络，为DSB的处理、细胞适应、细胞程序性死亡或衰老奠定基础。DSB在细胞中得到有效处理。然而，DSB与严重的生物效应有关，因为它们的处理与突变和染色体易位有关。本项目调查DSB处理设备如何支持这些效果。根据不同的原理，DSB有四条途径：同源重组修复(HRR)、规范非同源末端连接(c-NHEJ)、交替末端连接(ALT-EJ)和单链退火法(SSA)。令人惊讶的是，只有HRR能够忠实地恢复基因组，而所有剩余的途径，包括酶占优势的c-NHEJ，都能耐受/催化突变和易位。因此，途径的选择决定了基因组的改变是否伴随着DSB的处理。在这里，我们提出并测试了一个关于DSB修复途径选择的新假说，有望促进我们对DSB处理内在的逻辑和必要性的理解。核心问题是：细胞如何在c-NHEJ和HRR之间做出选择？我们提供的证据表明，在c-NHEJ的起始步骤中，Ku从DNA末端移位，并由DNA-PK自动磷酸化介导，改变了Ku蛋白的构象，并诱导了与Mre11的相互作用，从而触发了切除和HRR。我们表明，在某些条件下，超过50%的DSB可以被HRR处理，这使得这种机制与基因组完整性高度相关。一大套有希望的初步实验指导了三个工作包的开发，测试Ku和Mre11在体外和体内的相互作用，并分析它们与c-NHEJ-和HRR-装置的联系，以及检查点反应。建议的DSB处理装置的分子表征将促进我们对基因组不稳定来源的理解，并将加速制定改进辐射防护和辐射治疗的策略。