Mechanistic Characterization of the Replication Stress Response

复制压力响应的机制表征

基本信息

批准号：
10715216
负责人：
Richard Adeyemi
金额：
$ 44万
依托单位：
FRED HUTCHINSON CANCER CENTER
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-08-15 至 2028-05-31
项目状态：
未结题

项目摘要

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.

项目概要准确的 DNA 复制是控制每个生物体生存的基本过程。细胞 DNA 不断受到来自各种来源的攻击。细胞复制机器经常遇到并且非常容易受到阻碍其前进的代理的影响，从而导致复制错误、突变的发生以及各种形式的遗传不稳定性。因此其中，复制应激水平升高是各种疾病的特征标志状况。因此，从分子细节上理解不同的机制至关重要哪些细胞在复制应激过程中对受损的 DNA 做出反应并充分修复。虽然我们对 DNA 断裂如何修复了解很多，但我们的理解存在重大差距细胞如何充分应对复制压力，部分原因是缺乏遗传和生化工具来探测这些过程。为了进一步了解所涉及的流程在复制应激反应中，以及为了识别和表征全部基因该途径所需的，在我的博士后期间，我在多个细胞中进行了全基因组筛选用低剂量的复制应激诱导剂扰动后的细胞系。从这些我生成了一个新颖的数据集，其中包含尚未链接的多个基因基因组不稳定。几个新发现的基因与染色质反应有关，复制叉维持途径、核苷酸生物合成的调节等。的注意，我鉴定出了 Protexin 复合物，由单链 DNA 结合蛋白组成 SCAI 和 DNA 聚合酶 REV3。蛋白质对于维持基因组稳定性至关重要通过未知机制调节单链 DNA 积累。这些屏幕还揭示了 RNA 依赖性过程在复制应激反应中的显着作用，以前从未受到重视的新颖监管层。在我们的热门歌曲中，我们鉴定了几种新型 RNA 解旋酶和 RNA 结合因子，以及几种非编码 RNA 分子，证明了 RNA 依赖性过程和充分维持基因组稳定性。我的实验室将利用这一巨大的资源新发现的因素来表征新的基因组维护机制。调查中这些新颖的因素将使我们能够详细解读协调一致的多层修复暴露于 DNA 损伤时的反应和分叉恢复控制。我们将 (1) 描述复制应激后单链积累的机制。 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过程中的作用。完成这些研究项目将为我们带来重要且基本的新颖见解研究细胞基因组在面对破坏性侮辱时如何维持，让我们改进更详细和开放地了解癌症发病机制的机制为利用有缺陷的 DNA 修复过程治疗癌症开辟新途径。