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

摘要 DNA双链断裂(DSB)对基因组完整性和细胞生存构成严重威胁，并 癌症的驱动因素。非同源末端连接(NHEJ)负责修复这些断裂的大部分 在高等真核生物中。NHEJ突触复合体由核心因子Ku、DNA-PKcs、XRCC4/LigIV、 和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 处理并比对它们，以便通过XRCC4/LigIV复合体直接连接。通过利用单分子FRET (SmFRET)在非洲爪哇卵提取液中实时监测DNA末端突触 系统，我们的实验室最近表明，NHEJ的修复至少经历了两个不同的阶段。DNA末端 首先在末端相距超过10 nm的长距离复合体中突触。只有Ku和DNA- 需要PKC才能形成这种状态。接下来，在短距离连接之前，将DNA末端紧密对齐 复杂。向短程复合体的转变需要DNA-PKcs激酶活性和存在 XLF和XRCC4/LigIV。然而，LigIV的催化活性不是形成短程络合物所必需的。什么 推动这两个不同国家之间的过渡仍不清楚。 在这项建议中，我的目标是通过继续使用来确定XRCC4/LigIV在DNA末端突触中的作用 在单分子荧光实验中对非洲爪哇卵子提取系统进行了研究。以初步数据为基础 为了证明XLF的单个副本足以进行末端连接，我将确定XRCC4/LigIV的数量 和游离态的XRCC4以双链断裂的形式存在和作用。我将生成标记的XRCC4/LigIV和免费的 XRCC4构建了使用三色单分子成像直接确定每个DSB的拷贝数的结构。 此外，我将确定是否需要与XLF交互以保留或稳定XRCC4/LigIV或FREE XRCC4在短程复合体内。 观察到，需要LigIV，而不是它的催化活性，才能进展到短程络合物 提示LigIV在突触复合体组装中具有结构性作用。为了确定这一角色的基础，我将 生成一系列N端截断突变体，揭示LigIV结构域的最小要求 射程复合体编队。驱动短程复合体形成的相互作用是否涉及DNA或 LigIV获得DNA末端所需的基因目前尚不清楚。我将采用三色smFRET策略来 测量LigIV与DNA末端相互作用的时间，相对于短程复合体的形成。这些 调查结果有可能在实地产生重大影响，因为最关键的组成部分(S)的作用 NHEJ，XRCC4/LigIV，仍然定义不清。阐明XRCC4/LigIV的机制，以及更广泛的 NHEJ，将允许更好地了解疾病，并为新疗法的开发和 生物技术应用。

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.