DNA damage response and repair of a broken chromosome

项目摘要 我实验室由NIGMS资助的研究的总体目标是描述分子和细胞 细胞感知DNA损伤的存在并进行我们的修复的机制 染色体双链断裂(DSB)。使用发芽酵母作为模型系统，这是可能的 为了诱导在哺乳动物细胞中尚不可能实现的高度同步性的位点特异性DSB， 允许“体内生物化学”方法监测DSB修复和DNA的中间步骤 损坏信号。这项提案的三个焦点中的第一个调查了以下关键问题 DSB通过同源重组修复。同源供体序列是如何找到的 用来修理DSB的？如何在不同的步骤中容忍和修复不匹配 重组？细胞在修复过程中如何处理染色质，染色质是如何重新... 在维修完成后建立？基因编辑是如何使用单链的 DNA模板？第二个领域试图了解1000倍增长的基础是什么 在与DSB修复相关的突变中以及微同源如何用于修复依赖中 模板切换，创建类似于最近发生的事件的复杂染色体重排 在人类癌症中发现。第三个主要目标是了解DNA损伤是如何 检查点和DNA损伤诱导的自噬受到调控。我们希望确定：如何 DNA损伤反应会影响DSB修复吗？DNA损伤检查点是如何维护的 Mec1ATR的自动磷酸化是如何调节的？监管移交的基础是什么？ 在损坏响应和主轴组件检查点之间？最后，我们将确定 DNA损伤诱导自噬的靶点及其在细胞周期停滞中的作用 以响应哪怕是一个未修复的DSB。这些研究将提供新的见解和 在人类细胞中定义DSB修复和检查点信号的指南。

Project Summary The overall goal of NIGMS-funded research in my lab is to describe the molecular and cellular mechanisms by which cells sense the presence of DNA damage and carry our repair of chromosomal double-strand breaks (DSBs). Using budding yeast as a model system, it is possible to induce site-specific DSBs with a high degree of synchrony not yet possible in mammalian cells, allowing “in vivo biochemistry” approaches to monitor intermediate steps in DSB repair and DNA damage signaling. The first of three foci of this proposal investigates key questions concerning DSB repair by homologous recombination. How are homologous donor sequences found and used to repair a DSB? How are mismatches tolerated and repaired during different steps of recombination? How do cells deal with chromatin during repair and how is chromatin re- established after repair is complete? And how is gene editing accomplished using single-stranded DNA templates? The second area seeks to understand what is the basis of the 1000-fold increase in mutations associated with DSB repair and how microhomologies are used in repair-dependent template switching, creating complex chromosome rearrangements analogous to events recently found in human cancers. The third main objective is to understand how the DNA damage checkpoint and DNA damage-induced autophagy are regulated. We wish to determine: How does the DNA damage response affect DSB repair? How is the DNA damage checkpoint maintained and how is autophosphorylation of Mec1ATR regulated? What is the basis of a regulatory hand-off between the damage response and the spindle assembly checkpoint? Finally, we will determine the targets of DNA damage-induced autophagy and how autophagy contributes to cell cycle arrest in response to even a single unrepaired DSB. These studies will provide new insights and guidance in defining the DSB repair and checkpoint signaling in human cells.