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

 描述(申请人提供)：该项目使用新的单分子和整体生物化学方法相结合的方法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期间，DNA末端首先被Ku70/80异二聚体识别和结合，而Ku70/80异二聚体又招募了许多其他负责桥接末端的因子，处理它们进行连接，并最终共价连接它们。目前尚不清楚这些NHEJ因子如何将DNA末端拴在突触复合体中，以及末端突触、加工和连接是如何协调的。我们将使用单分子FRET方法在非洲爪哇卵提取液中实时观察NHEJ。这些分析将使我们能够表征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更深入地了解双链断裂的修复，例如由放射治疗引起的或被新兴的基因组编辑技术利用的双链断裂的修复。目的1)确定DNA末端在非同源末端连接过程中是如何连接的必须有效地连接断裂的DNA末端，以防止它们扩散开来并连接到错误的配对。在体外重建实验中，几乎所有的核心NHEJ因子都参与了末端拴系，但在生理条件下不同的因子在DNA末端突触中起什么作用尚不清楚。使用我们在我的实验室开发的单分子FRET分析，我们将实时跟踪DNA末端的突触，并表征负责它们系系的NHEJ因子。目的2)确定NHEJ机制在末端连接过程中的组成如何变化。为此，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体的组成。首先，我们将开发大量的生化方法来捕获我们的单分子实验所识别的不同的突触中间体，并用质谱仪分析它们的组成。接下来，我们将进行单分子成像实验，以表征XRCC4-LIG4和XLF与不同突触复合体的关联，并确定这种关联如何受到DNA-PK活性的调节。目的3)阐明在NHEJ过程中末端处理是如何调节的，以保持其保真度，化学损伤或不相容的DNA末端必须经过处理才能连接。目前尚不清楚NHEJ机制是如何调节过程以将突变降至最低的。最近对NHEJ连接的深度测序支持的一种可能性是，在任何处理之前，尝试在突触复合体内进行连接。为此，我们将测试形成突触复合体所需的因子是否也需要在不同类型的DNA末端启动处理，并使用单分子成像来直接可视化具有不相容末端的底物的处理和尝试连接。

 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.