The of Role DNA LigIV and Its Accessory Factors in the NHEJ Synaptic Complex

DNA LigIV及其辅助因子在NHEJ突触复合体中的作用

• 批准号: 9770538
• 负责人: Sean Michael Carney
• 金额: $ 6.37万
• 依托单位: HARVARD MEDICAL SCHOOL
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2020-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9770538
• 关键词: Binding Sites; Biotechnology; Cell Survival; Cells; Chromosome Pairing; Color; Complex; DNA; DNA Double Strand Break; DNA Ligation; DNA Repair Pathway; DNA-PKcs; Data; Development; Disease; Double Strand Break Repair; Eukaryota; Event; Filament; Fluorescence; Fluorescence Resonance Energy Transfer; Goals; Immune system; Knowledge; Label; Laboratories; Ligase; Ligation; Malignant Neoplasms; Measures; Methods; Modeling; Monitor; N-terminal; Nonhomologous DNA End Joining; Phosphotransferases; Physiological; Process; Proteins; Role; Series; Structure; Synapses; System; Systems Development; Technology; Testing; Time; XRCC4 gene; Xenopus; cytotoxic; dimer; egg; experimental study; fluorescence imaging; genome integrity; molecular imaging; mutant; novel therapeutics; recruit; repaired; single molecule; single-molecule FRET; stoichiometry

Abstract DNA Double strand breaks (DSBs) pose a serious threat to genomic integrity and cell survival, and are drivers of cancer. Non-homologous end joining (NHEJ) is responsible for repairing the majority of these breaks in higher eukaryotes. The NHEJ synaptic complex consists of the core factors Ku, DNA-PKcs, XRCC4/LigIV, and XLF. Together these repair proteins must first recognize the DSB, tether the DNA ends together, and then process and align them for direct ligation by the XRCC4/LigIV complex. By utilizing single-molecule FRET (smFRET) to monitor DNA end synapsis in real time within the context of the physiological Xenopus egg extract system, our lab has recently shown that repair by NHEJ proceeds through at least two distinct stages. DNA ends are first synapsed in a Long Range Complex where the ends are more than 10 nm apart. Only Ku and DNA- PKcs are required to form this state. Next, the DNA ends are closely aligned prior to ligation in a Short Range Complex. The transition to the Short Range Complex requires DNA-PKcs kinase activity and the presence of XLF and XRCC4/LigIV. However, LigIV’s catalytic activity is not required to form the Short Range Complex. What drives the transition between these two distinct states remains unclear. In this proposal, I aim to determine the role of XRCC4/LigIV in DNA end synapsis through continued use of the Xenopus egg extract system in single-molecule fluorescence experiments. Building on preliminary data demonstrating that a single copy of XLF is sufficient for end joining, I will determine the number of XRCC4/LigIV and free XRCC4 present and acting at a double strand breaks. I will generate labeled XRCC4/LigIV and free XRCC4 constructs to directly determine the copy number of each at DSBs using 3-color single-molecule imaging. Additionally, I will determine whether interaction with XLF is required to retain or stabilize XRCC4/LigIV or free XRCC4 within the Short Range Complex. The observation that LigIV, but not its catalytic activity, is needed to progress to the Short Range Complex suggests that LigIV has a structural role in synaptic complex assembly. To determine the basis of this role, I will generate a series of N-terminal truncation mutants, and reveal the minimal LigIV domain requirements for Short Range Complex formation. Whether the interactions that drive Short Range Complex formation involve DNA or are required for LigIV to gain access to the DNA ends is unclear. I will employ a 3-color smFRET strategy to measure when LigIV interacts with the DNA ends relative to the formation of the Short Range Complex. These findings will have the potential for significant impact in the field, as the role(s) of the most critical component of NHEJ, XRCC4/LigIV, remains poorly defined. Elucidating the mechanism of XRCC4/LigIV, and more broadly NHEJ, will allow for a better understanding of disease and inform the development of new therapies and biotechnology applications.

摘要 DNA双链断裂（DSB）对基因组完整性和细胞存活构成严重威胁， 癌症的司机非同源末端连接（NHEJ）负责修复大多数这些断裂 在高等真核生物中。NHEJ突触复合体由核心因子Ku、DNA-PKcs、XRCC 4/LigIV组成， 还有XLF。这些修复蛋白必须首先识别DSB，将DNA末端连接在一起，然后 处理并排列它们，用于通过XRCC 4/LigIV复合物直接连接。通过利用单分子FRET （smFRET）监测DNA末端突触在真实的时间内的背景下的生理爪蟾卵提取物 系统，我们的实验室最近表明，修复NHEJ进行通过至少两个不同的阶段。DNA末端 首先在长距离复合体中突触，其中末端相距超过10 nm。只有Ku和DNA- 需要PKC来形成这种状态。接下来，在短范围内连接之前，将DNA末端紧密对齐。 复杂.向短程复合物的转变需要DNA-PKcs激酶活性和 XLF和XRCC 4/LigIV。然而，LigIV的催化活性不是形成短程复合物所必需的。什么 这两种不同状态之间的转换驱动力仍不清楚。 在这个提议中，我的目标是通过继续使用XRCC 4/LigIV来确定XRCC 4/LigIV在DNA末端突触中的作用。 的爪蟾卵提取物系统在单分子荧光实验。根据初步数据 为了证明XLF的单个拷贝足以进行末端连接，我将确定XRCC 4/LigIV的数目， 和存在并作用于双链断裂处的游离XRCC 4。我将生成标记的XRCC 4/LigIV和游离的 XRCC 4构建体，以使用3色单分子成像直接确定每个在DSB处的拷贝数。 此外，我将确定是否需要与XLF的相互作用来保留或稳定XRCC 4/LigIV或游离XRCC 4/LigIV。 短程复合物中的XRCC 4。 观察到LigIV，而不是其催化活性，需要进展到短程复合物 表明LigIV在突触复合体组装中具有结构作用。为了确定这一角色的基础，我将 产生了一系列的N-末端截短突变体，并揭示了短的最小LigIV结构域的要求， 靶场综合体编队。驱动短程复合物形成的相互作用是否涉及DNA或 LigIV进入DNA末端所需的条件尚不清楚。我将采用3色smFRET策略， 测量相对于短程复合物的形成，LigIV何时与DNA末端相互作用。这些 调查结果将有可能在实地产生重大影响，因为调查结果中最关键的组成部分的作用 NHEJ，XRCC 4/LigIV，仍然定义不清。阐明XRCC 4/LigIV的机制，更广泛地 NHEJ将允许更好地了解疾病，并为新疗法的开发提供信息， 生物技术应用。