Investigating DNA end-processing during non-homologous end joining

研究非同源末端连接过程中的 DNA 末端加工

基本信息

批准号：
10463957
负责人：
Brandon C Case
金额：
$ 6.76万
依托单位：
HARVARD MEDICAL SCHOOL
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-07-21 至 2023-07-20
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10463957
关键词：
Affinity Back Binding Biological Assay Cell Cycle Cell Survival Cells Chemicals Chromosomal Rearrangement Chromosomal translocation Chromosome Pairing Color Complex Controlled Environment DNA DNA Damage DNA Double Strand Break DNA Repair DNA biosynthesis DNA lesion DNA-PKcs DNA-dependent protein kinase Data Dependence Enzymes Eukaryota Fluorescence Genetic Genome Stability Genomics Goals Human Immune system Knock-out Label Laboratories Lead Left Ligation Malignant Neoplasms Mass Spectrum Analysis Mediating Methods Modeling Modernization Molecular Monitor Mutagenesis Nonhomologous DNA End Joining Nucleotides Outcome Pathway interactions Phosphorylation Phosphotransferases Physiological Play Polymerase Post-Translational Protein Processing Process Proteins Proteome Radiation Reaction Recombinants Regulation Resolution Risk Role Source Surgical Flaps System Systems Development Techniques Testing Time V(D)J Recombination Validation Work XRCC4 gene Xenopus crosslink cytotoxic design ds-DNA egg experimental study fluorescence imaging genetic information genome editing genome integrity insertion/deletion mutation molecular imaging nuclease prevent protein protein interaction recruit repaired single molecule single-molecule FRET tool tumorigenesis

项目摘要

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.

抽象的 DNA 中的双链断裂 (DSB) 对基因组完整性和细胞存活构成严重威胁。断裂由内源性和外源性来源引起并促进非同源末端的发生。连接（NHEJ）在整个细胞周期中都很活跃，负责修复较高级的大多数 DSB。我们实验室之前的工作确定了末端连接反应中的两个关键中间体，称为长程复合物 (LRC) 和短程复合物 (SRC) Ku 与 DSB 快速结合，然后进行招募。 DNA-PKcs 介导 LRC 形成，其中两个 dsDNA 末端连接在一起但不紧密对齐随后的 DNA-PK 激酶活性以及 XRCC4/LigIV 和 XLF 的结合促进了转变。然而，从 LRC 到 SRC，相对的 DNA 末端经常被连接。化学损坏，因此不适合连接 NHEJ 机器使用大量末端。我们的实验室已经证明了可以纠正这种损坏的加工因素，从而实现端部连接。处理主要限于 SRC，它优先考虑连接而不是处理，并且还最大限度地减少了此外，我们实验室的数据表明，处理的程度达到了连接所需的程度。末端处理有利于损伤校正（即去/磷酸化）而不是容易出错的（即聚合酶和核酸酶）维持基因组稳定性的加工因子，SRC 有利于减少诱变。修复情况仍不清楚。在这个提案中，我的目标是了解两个末端处理 NHEJ 因素 - PNKP（损伤校正）和聚合酶 λ（容易出错）- 利用非洲爪蟾卵提取物系统组合来创建可连接末端基于证明处理的初步数据的单分子荧光实验。如果因子能够接触到 SRC 内的 DNA 末端，我会问如何调节因子招募以及如何为此，我生成了荧光标记的 NHEJ。使用三色 FRET 单分子成像直接监测 SRC 募集的处理因素。此外，我将确定损害校正和易错因素之间是否存在竞争或招募解释了如何实现最少的处理。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性的丧失。尽管 NHEJ 涉及许多核酸酶，但许多核酸酶的生理相关数据很少。为了识别在 NHEJ 期间发挥作用的核酸酶，我开发了一种下拉瓣的交联测定法。使用鸡蛋提取物系统通过质谱分析相关蛋白质的活性。然后，将通过在耗尽和回加测定中测试核酸酶的活性来验证核酸酶。将增进我们对 NHEJ 如何调节容易出错的末端处理的机制理解，这可能为基因组编辑应用中调节修复保真度的努力提供信息。