Defining the mechanism and specificity of the 53BP1-Rev7 non-homologous end joining pathway in immunity and oncogenesis

该研究项目的主要目的是完善我们对53BP1通路的理解：这是一个鲜为人知的脊椎动物非同源末端连接（NHEJ） DNA修复系统的亚通路，在人类适应性免疫应答中起着至关重要的作用，但也驱动了BRCA1肿瘤抑制基因错误拷贝患者乳腺癌、卵巢癌和其他肿瘤的发生和进展。该项目的一个关键目标是挑战这一途径的分子功能的广泛接受的范式。我们将着手检验一个假说，该假说为这一通路的活动提供了另一种解释。也就是说，在缺乏53BP1通路的细胞中，在DNA双链断裂（DSB）位点检测到的单链DNA富集，并不像人们普遍认为的那样代表了保护DNA末端不被切除的失败产物，而是代表了当通路失败时无法克服的结构化DNA修复中间物/底物。如果正确的话，我们计划设计的转基因小鼠将由于无法通过53bp1依赖的NHEJ有效地产生抗体多样性而产生免疫缺陷。然而，来自这些动物的细胞在缺乏BRCA1活性的情况下能够存活并相对健康地分裂，BRCA1是维持正常细胞增殖和维持基因组稳定性所必需的蛋白质。这两种情况都将为我们提供有价值的生物学工具，使我们能够在与人类健康和疾病直接相关的生物学背景下，将机制归因于53BP1 DNA修复系统。其他不同但互补的实验将检验我们模型的次要预测，包括一个可能解释53bp1依赖的DNA修复系统的环境特异性，从而将其免疫系统功能与其在癌症中的强致癌活性联系起来的实验。

The major aim of this research project is to refine our understanding of the 53BP1 pathway: a poorly understood sub-pathway of the vertebrate non-homologous end joining (NHEJ) DNA repair system that plays a vital role in the human adaptive immune response, yet also drives the initiation and progression of breast, ovarian and other tumours in patients harbouring a faulty copy of the BRCA1 tumour suppressor gene. A key objective of the project is to challenge a widely accepted paradigm for the molecular function of this pathway. We will set out to test a hypothesis that proposes an alternative explanation for this pathway's activity. Namely, that the single-stranded DNA enrichments detected at DNA double-strand break (DSB) sites in cells deficient for the 53BP1 pathway, do not represent product of a failure to protect DNA ends from resection as is widely perceived, but instead represents a structured DNA repair intermediate/substrate that cannot be overcome when the pathway fails. If correct, the genetically modified mice we plan to engineer will be immune-deficicient as a result of an inability to efficiently generate antibody diversity via 53BP1-dependent NHEJ. Nevertheless, cells from these animals will be able to survive and divide relatively healthily in the absence of the activity of BRCA1, a protein normally essential for sustaining normal cell proliferation and the maintenance of genome stability. Both scenarios will provide us with valuable biological tools with which we will attribute mechanism to the 53BP1 DNA repair system, in biological contexts directly related to human health and disease. Other distinct, yet complementary experiments will examine secondary predictions of our model, including one that may explain the context specificity of the 53BP1-dependent DNA repair system, thereby linking its immune system functions to its potent oncogenic activities in cancer.