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

电离辐射（IR）通过诱导DNA双链断裂（DSBs）产生不良影响，如细胞杀伤或致癌。DSB是引起细胞“DNA损伤反应（DDR）”的严重病变：一个广泛改变细胞代谢的信号转导通路网络，为DSB处理、细胞适应、程序性细胞死亡或衰老奠定了基础。dsb在细胞中被有效地处理。然而，dsb与严重的生物效应有关，因为它们的加工与突变和染色体易位有关。本项目研究DSB处理装置如何支持这些影响。四种不同原理的处理dsb的途径：同源重组修复（HRR），规范非同源末端连接（c-NHEJ），替代末端连接（alt-EJ）和单链退火（SSA）。令人惊讶的是，只有HRR能够忠实地恢复基因组，而所有其他途径，包括酶上占主导地位的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改变是否伴随DSB加工。在这里，我们提出并验证了一个关于DSB修复途径选择的新假设，有望促进我们对DSB加工内在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的初始阶段，Ku从DNA端转移并通过DNA- pk自磷酸化介导，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可能被HRR处理，这使得这种机制与基因组完整性高度相关。大量有前景的初步实验指导了三个工作包的开发，这些工作包在体外和体内测试Ku和Mre11之间的相互作用，并分析它们与c-NHEJ-和hrr -装置以及检查点反应的联系。提出的DSB处理装置的分子表征将促进我们对基因组不稳定性来源的理解，并将加速开发改善放射保护和放射治疗的策略。

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.