Understanding the mechanistic role of genome stability pathways in regulating cell homeostasis

项目概要/摘要 为了保持基因组的完整性和维持体内平衡，我们体内的细胞必须有效地对这两者做出反应。 DNA损伤的外源性和内源性来源。细胞DNA损伤如何导致人类疾病， 包括神经系统疾病和癌症，是一个基本的研究领域。在过去的13年里，我 实验室一直专注于与基因组不稳定性的机制基础相关的研究。这个MIRA的目标 应用程序是解决我们对基因组稳定性途径的理解以及它们如何 在分裂细胞与非分裂细胞中有区别地使用，以适当维持细胞内稳态。通过 细胞表面受体酪氨酸激酶（RTK）将外部信息从细胞外周传递到细胞核， 通过PI 3-激酶（PI 3 K）-AKT信号传导对生长因子作出反应，以调节基因表达，从而促进 生长和/或存活。同样，DNA损伤威胁基因组的完整性， 增强DNA损伤反应（DDR）信号传导以帮助DNA修复和细胞周期检查点。为我们的研究 计划，我们将解决有关如何关键DDR因素有助于细胞外生长的独特机制 分裂和非分裂细胞中的信号串扰因素。为了建立这些机制，我们将利用 一系列创新的实验方法，包括全基因组测序，邻近连接蛋白质组学， 超分辨率显微镜和活细胞中的单分子跟踪。我们将检验DDR 应被视为一个更广泛的，应激反应网络连接核和细胞质效应，以维持 通过与生长因子信号通路交叉来实现生理稳态。这是如何实现 机械地将是本申请的主要焦点。第二个项目涉及如何解决遗传毒性 分裂细胞中的应激影响DNA复制动力学，并阐明调节 复制分叉恢复。基于我们称为冈崎片段测序（OK-seq）的创新技术，我们 能够直接量化整个过程中特定站点处复制叉启动和终止的效率 基因组使用这种技术，我们将扩大我们的分析，以了解如何遗传毒性的侮辱和DNA 使用全基因组分析，修复缺陷导致位点特异性复制叉介导的DNA断裂。 破译与复制相关的基因组不稳定性的机制可能会提供新的 有针对性的癌症治疗的途径。

PROJECT SUMMARY/ABSTRACT To preserve genomic integrity and maintain homeostasis, cells in our body must effectively respond to both exogenous and endogenous sources of DNA damage. How cellular DNA damage contribute to human disease, including neurological disorders and cancer, is a fundamental area of research. For the past thirteen years, my lab has been focused on studies related to the mechanistic basis of genomic instability. The goal of this MIRA application is to address critical gaps in our understanding of genome stability pathways and how they are differentially utilized in dividing versus non-dividing cells for the proper maintenance of cellular homeostasis. By relaying external information from the cell periphery to the nucleus, cell surface receptor tyrosine kinases (RTKs) respond to growth factors via PI3-kinase (PI3K)-AKT signaling to regulate gene expression and thereby promote growth and/or survival. Similarly, DNA damage threatens genome integrity and upon detection within the nucleus elicits DNA damage response (DDR) signaling to aid in DNA repair and cell cycle checkpoints. For our research program, we will address unique mechanisms related to how key DDR factors contribute to extracellular growth factor signaling crosstalk in dividing and non-dividing cells. To establish these mechanisms, we will utilize an array of innovative experimental approaches including genome-wide sequencing, proximity ligation proteomics, super-resolution microscopy and single-molecule tracking in live cells. We will test the hypothesis that the DDR should be viewed as a broader, stress-responsive network linking nuclear and cytoplasmic effectors to maintain physiological homeostasis through intersecting with growth factor signaling pathways. How this is achieved mechanistically will be a major focus of this application. The second project involves addressing how genotoxic stress in dividing cells impacts DNA replication dynamics and to elucidate novel molecular players that regulate replication fork recovery. Based on our innovative technique called Okazaki fragment sequencing (OK-seq), we are able to directly quantify the efficiency of replication fork initiation and termination at specific sites throughout the genome. Using this technique, we will expand our analysis to understand how genotoxic insults and DNA repair deficiencies contribute to site-specific replication fork-mediated DNA breaks using genome-wide analysis. Deciphering the mechanisms that contribute to replication-associated genomic instability may provide new avenues for targeted cancer treatment.