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

这项研究的主要目的是加深我们对53BP1途径的理解：这是脊椎动物非同源末端连接(NHEJ)DNA修复系统中一个知之甚少的子途径，在人类适应性免疫反应中发挥着至关重要的作用，但也推动了BRCA1肿瘤抑制基因拷贝错误的患者乳腺癌、卵巢和其他肿瘤的发生和发展。该项目的一个关键目标是挑战这一途径的分子功能的一个广泛接受的范例。我们将着手测试一个假说，该假说为这一途径的活动提出了另一种解释。也就是说，在53BP1途径缺失的细胞中在DNA双链断裂(DSB)位置检测到的单链DNA富集物，并不是人们普遍认为的未能保护DNA末端免受切除的产物，而是一种结构化的DNA修复中间/底物，当该途径失败时无法克服。如果正确，我们计划设计的转基因小鼠将是免疫缺陷的，因为无法通过依赖53BP1的NHEJ有效地产生抗体多样性。然而，在缺乏BRCA1活性的情况下，这些动物的细胞将能够相对健康地存活和分裂。BRCA1通常是维持正常细胞增殖和维持基因组稳定所必需的蛋白质。这两种情况都将为我们提供宝贵的生物学工具，利用这些工具，我们将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查我们模型的二级预测，包括一个可能解释53BP1依赖的DNA修复系统的上下文特异性，从而将其免疫系统功能与其在癌症中的强大致癌活动联系起来的实验。

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.