Investigating DNA end-processing during non-homologous end joining

摘要 DNA双链断裂（DSB）对基因组的完整性和细胞的生存构成了严重的威胁。这些 断裂来自内源性和外源性来源，并促进肿瘤发生。非同源末端 连接（NHEJ）在整个细胞周期中是活跃的，并负责修复大多数较高水平的DSB。 真核生物我们实验室以前的工作确定了末端连接反应中的两个关键中间体，称为 长程复合波（LRC）和短程复合波（SRC）。Ku与DSB的快速结合，随后进行募集 DNA-PKcs介导LRC的形成，其中两个dsDNA末端被拴在一起但不紧密对齐 用于结扎。随后的DNA-PK激酶活性以及XRCC 4/LigIV和XLF的结合促进了这种转变。 从LRC到SRC，其中相对的DNA末端准备被连接。然而，DNA末端通常 化学损伤，因此不适合结扎。NHEJ机器利用大量的终端 处理因素来校正这种能够进行端部连接的损坏。我们的实验室已经证明了 处理在很大程度上限于SRC，SRC使连接优先于处理，并且还使连接最小化。 加工到能够连接所必需的程度。此外，我们实验室的数据表明， 末端加工有利于损伤校正（即去/磷酸化）而不是易错（即聚合酶和 核酸酶）加工因子以维持基因组稳定性。SRC倾向于降低致突变性的方法 修复尚不清楚。 在这个建议中，我的目的是了解如何两个端处理NHEJ因素- PNKP（损伤校正） 和聚合酶λ（易错）-起作用以利用组合的爪蟾卵提取物系统产生可连接末端， 单分子荧光实验。根据初步数据， 因子在SRC内获得DNA末端，我将询问因子募集是如何调节的，以及 保持了对DNA末端的访问的层次结构。为此，我用荧光标记的NHEJ 使用三色FRET单分子成像直接监测SRC的募集。 此外，我将确定是否竞争或招聘损害纠正和错误倾向的因素， 说明如何实现最小化处理。 核酸酶是最具致突变性的末端加工因子，因为它们的活性总是导致遗传修饰的丧失。 信息.虽然许多核酸酶与NHEJ有关，但对于许多核酸酶，几乎没有生理学相关的数据。 的索赔。为了鉴定在NHEJ中起作用的核酸酶，我开发了一种用于下拉瓣的交联测定法， 相关的蛋白质，用于使用蛋提取物系统通过质谱进行鉴定。这些活动 然后通过在消耗和添加回测定中测试核酸酶的活性来验证核酸酶。总的来说，这项提案 将推进我们对NHEJ如何调节易出错的末端处理的机械理解， 为在基因组编辑应用中调节修复保真度的努力提供信息。

Abstract Double strand breaks (DSBs) in DNA pose a serious threat to genomic integrity and cell survival. These breaks arise from endogenous and exogenous sources and promote tumorigenesis. Non-homologous end joining (NHEJ) is active throughout the cell cycle and is responsible for repairing the majority of DSBs in higher eukaryotes. Previous work from our lab identified two key intermediates in the end joining reaction, termed the long-range complex (LRC) and short-range complex (SRC). Rapid binding of Ku to DSBs followed by recruitment of DNA-PKcs mediates LRC formation where the two dsDNA ends are tethered together but not aligned closely for ligation. Subsequent DNA-PK kinase activity and binding of XRCC4/LigIV and XLF promote the transition from the LRC to the SRC, where opposing DNA ends are poised to be ligated. DNA ends, however, are frequently chemically damaged and thus incompatible for ligation. The NHEJ machinery utilizes a vast array of end processing factors to correct this damage which enable end joining. Our lab has demonstrated that end processing is largely restricted to the SRC which prioritizes ligation over processing and also minimizes the extent of processing to what is necessary to enable ligation. Moreover, data from our laboratory suggests that end processing favors damage-correction (i.e. de/phosphorylation) over error-prone (i.e. polymerases and nucleases) processing factors to maintain genomic stability. The means by which the SRC favors less mutagenic repair remain unclear. In this proposal, I aim to understand how two end processing NHEJ factors - PNKP (damage-correction) and polymerase λ (error-prone) - act to create ligatable ends utilizing a Xenopus egg extract system combined with single-molecule fluorescence experiments. Building on preliminary data demonstrating that processing factors gain access to DNA ends within the SRC, I will ask how factor recruitment is regulated and how a hierarchy of access to DNA ends is maintained. To this end, I have generated fluorescently labeled NHEJ processing factors to directly monitor recruitment to the SRC using three-color FRET single-molecule imaging. Additionally, I will determine if competition between or recruitment of damage-correction and error-prone factors explains how minimal processing is achieved. Nucleases are the most mutagenic end processing factors as their activity always leads to loss of genetic information. Though many nucleases are implicated in NHEJ, there is little physiologically relevant data for many of the claims. To identify nucleases acting during NHEJ, I developed a crosslinking assay to pull-down flap associated proteins for identification via mass spectroscopy using the egg extract system. The activity of these nucleases will then be validated by testing their activity in a depletion and add-back assay. Overall, this proposal will advance our mechanistic understanding of how NHEJ regulates error-prone end processing which may inform efforts to modulate the fidelity of repair in genome editing applications.