Visualizing DNA break repair: single-molecule studies of non-homologous end joining

 描述（由申请人提供）：本项目使用新型单分子和批量生物化学方法的组合来研究非同源末端连接（NHEJ），这是人类细胞中DNA双链断裂修复的主要机制。在NHEJ过程中，DNA末端首先被Ku 70/80异二聚体识别和结合，Ku 70/80异二聚体又招募了许多其他因子，这些因子负责桥接末端，加工它们以进行连接并最终共价连接它们。目前还不清楚这些NHEJ因子如何将DNA末端拴在突触复合物中，以及末端突触、加工和连接如何协调。我们将使用单分子FRET方法观察非洲爪蟾卵提取物中真实的NHEJ。这些测定将使我们能够表征NHEJ机制中以前无法检测到的中间步骤。以下目标的完成将使我们对双链断裂的修复有更多的分子见解，例如由放射治疗诱导或新兴基因组编辑技术利用的双链断裂。目的1）确定在非同源末端连接期间DNA末端如何被拴系断裂的DNA末端必须被有效地拴系以防止它们扩散开并连接到错误的伴侣。在体外重建实验中，几乎每一个核心NHEJ因子都参与了末端束缚，但仍不清楚不同因子在生理条件下DNA末端的突触中起什么作用。使用单分子FRET分析，我们已经在我的实验室开发，我们将遵循突触的DNA末端在真实的时间和表征NHEJ的因素负责他们的束缚。目的2）确定NHEJ机制在末端连接过程中的组成如何变化在这个目的中，我们将确定不同突触中间体的组成，并研究DNA-PK活性如何调节突触复合物的组成。首先，我们将开发大量的生物化学方法来捕获我们的单分子实验所确定的独特的突触中间体，我们将通过质谱分析它们的组成。接下来，我们将进行单分子成像实验，以表征XRCC 4-LIG 4和XLF与不同突触复合物的关联，并确定这种关联如何受DNA-PK活性的调节。目的3）阐明在NHEJ期间如何调节末端加工以保持保真度，必须加工化学损伤或以其他方式不相容的DNA末端以允许连接。目前尚不清楚NHEJ机制如何调节加工以最大限度地减少突变。最近对NHEJ连接的深度测序支持的一种可能性是，在任何加工之前，在突触复合物内尝试连接。在ths的目标，我们将测试是否突触复合物形成所需的因素也需要启动处理在不同类型的DNA末端，并使用单分子成像直接可视化的处理和尝试连接基板与不兼容的结束。

 DESCRIPTION (provided by applicant): This project uses a combination of novel single-molecule and bulk biochemical methods to study non- homologous end joining (NHEJ), the predominant mechanism of DNA double strand break repair in human cells. During NHEJ, DNA ends are first recognized and bound by the Ku70/80 heterodimer, which, in turn, recruits a number of other factors that are responsible for bridging the ends, processing them for ligation and ultimately covalently joining them. It remains unclear how these NHEJ factors tether DNA ends in a synaptic complex and how end synapsis, processing, and ligation are coordinated. We will use single-molecule FRET approaches to observe NHEJ in real time in Xenopus egg extracts. These assays will allow us to characterize previously undetectable intermediate steps in the NHEJ mechanism. The completion of the following aims will give us increased molecular insight into the repair of double strand breaks, such as those induced by radiation therapy or exploited by emerging genome editing technologies. Aim 1) Determine how DNA ends are tethered during non-homologous end joining Broken DNA ends must be tethered efficiently to prevent them from diffusing apart and joining to the wrong partner. In vitro reconstitution experiments have implicated nearly every core NHEJ factor in end tethering, yet it remains unclear what role different factors play in the synapsis of DNA ends under physiological conditions. Using single-molecule FRET assays that we have developed in my lab, we will follow the synapsis of DNA ends in real time and characterize the NHEJ factors responsible for their tethering. Aim 2) Determine how the composition of the NHEJ machinery changes during end joining in this aim, we will determine the composition of different synaptic intermediates and investigate how DNA-PK activity regulates the composition of the synaptic complex. First, we will develop bulk biochemical methods to trap the distinct synaptic intermediates identified by our single-molecule experiments, and we will analyze their composition by mass spectrometry. Next, we will carry out single-molecule imaging experiments to characterize the association of XRCC4-LIG4 and XLF with different synaptic complexes and determine how this association is regulated by DNA-PK activity. Aim 3) Elucidate how end processing is regulated during NHEJ to maintain fidelity DNA ends that are chemically damaged or otherwise incompatible must be processed to permit ligation. It remains unclear how the NHEJ machinery regulates processing so as to minimize mutations. One possibility, supported by recent deep-sequencing of NHEJ junctions, is that ligation is attempted within the synaptic complex prior to any processing. In ths aim, we will test whether factors that are required for synaptic complex formation are also required to initiate processing at different types of DNA ends and use single-molecule imaging to directly visualize the processing and attempted ligation of substrates with incompatible ends.