Mechanistic Characterization of the Replication Stress Response

项目总结 准确的DNA复制是控制每个有机体生存的基本过程。 细胞DNA不断受到各种来源的攻击。细胞复制机制 经常遇到并严重容易受到阻碍其发展的代理的攻击，导致 复制错误、突变的发展和各种形式的遗传不稳定性。结果 其中，复制压力水平的升高是各种疾病的特征特征 条件。因此，在分子细节上理解不同的机制是至关重要的 在复制应激期间，哪些细胞对受损的DNA做出反应并进行适当的修复。虽然 关于DNA断裂是如何修复的，我们知道很多，但我们的理解存在很大差距 细胞如何对复制压力做出充分反应，部分原因是缺乏遗传和 生化工具来探测这些过程。进一步深入了解所涉及的过程 在复制应激反应中，为了识别和表征全套基因 在我的博士后期间，我在多个细胞中进行了全基因组筛选 在低剂量复制应激诱导剂的干扰下产生的线条。从这些 Screens I生成了一个新的数据集，其中包括多个尚未关联的基因 基因组不稳定。几个新发现的基因与染色质反应有关， 复制分叉维持途径，核苷酸生物合成的调节等。的 注意，我发现了Protex in复合体，由单链DNA结合蛋白组成 SCAI和DNA聚合酶Rev3。Protex in对维持基因组的不稳定性至关重要 通过未知的机制调节单链DNA的积累。这些屏幕 还揭示了RNA依赖过程在复制应激反应中的显著作用， 新奇的一层监管，这是以前没有人欣赏过的。在我们最热门的歌曲中，我们 确定了几个新的RNA解旋酶和RNA结合因子，以及几个非编码 RNA分子，展示了依赖RNA的过程和 充分维护基因组的稳定性。我的实验室将利用这一巨大的资源 新确定的因素，以表征新的基因组维持机制。调查 这些新颖的因素将使我们能够详细地破译协同的、多层次的修复 暴露于DNA损伤时的反应和叉子修复控制。我们将(1)描述 复制胁迫后单链积累的机制。2)识别 RNA修饰酶在复制应激反应中发挥作用的机制，以及 3)阐明非编码RNA基因在复制应激反应中的作用。 这些研究项目的完成将给我们带来重要的和基本的新见解 细胞基因组是如何在破坏性的侮辱面前保持的，请赐予我们改进 对癌症发病机制的更详细和更开放的理解 为在癌症治疗中利用有缺陷的DNA修复过程开辟了新的途径。

PROJECT SUMMARY Accurate DNA replication is a fundamental process that governs the survival of every organism. Cellular DNA is under constant assault from various sources. The cellular replication machinery frequently encounters and is severely vulnerable to agents that stall its advancement, leading to replicative errors, development of mutations, and various forms of genetic instability. As a result of this, elevated levels of replication stress is a characteristic hallmark of various disease conditions. It is therefore critical to understand in molecular detail the varied mechanisms by which cells respond to and adequately repair damaged DNA during replication stress. Although we know a lot about how breaks in DNA are repaired, there is a major gap in our understanding of how cells adequately respond to replication stress in part due to the lack of genetic and biochemical tools to probe these processes. To gain further insights into the processes involved in the replication stress response, and in order to identify and characterize the panoply of genes required for this pathway, during my postdoc I performed whole genome screens in multiple cell lines following perturbations with low doses of replication stress-inducing agents. From these screens I generated a novel dataset that includes multiple genes that have yet to be linked with genome instability. Several newly identified genes were linked to chromatin responses, replication fork maintenance pathways, regulation of nucleotide biosynthesis and others. Of note, I identified the Protexin complex, consisting of the single stranded DNA binding protein SCAI and the DNA polymerase REV3. Protexin was critical for maintaining genomic instability by regulating single stranded DNA accumulation through unknown mechanisms. These screens also revealed a striking role for RNA dependent processes in the replication stress response, a novel layer of regulation that had not been appreciated before now. Among our top hits, we identified several novel RNA helicases and RNA-binding factors, as well as several non-coding RNA molecules, demonstrating a crucial, intimate link between RNA-dependent processes and adequate maintenance of genome stability. My lab will take advantage of this vast resource of newly identified factors to characterize novel genome maintenance mechanisms. Investigating these novel factors will allow us to decipher in detail the concerted, multi-layered repair response and fork restoration control upon exposure to DNA damage. We will (1) characterize the mechanism of single stranded accumulation following replication stress. 2) Identify mechanisms by which RNA-modifying enzymes function in the replication stress response, and 3) elucidate roles for non-coding RNA genes during the replication stress Response. Completion of these research projects will grant us significant and fundamental novel insights into how cellular genomes are maintained in the face of damaging insults, grant us improved mechanistic understanding of the principles of cancer pathogenesis in greater detail and open up novel avenues for exploiting defective DNA repair processes in the treatment of cancers.