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

项目摘要/摘要 为了保持基因组的完整性和动态平衡，我们体内的细胞必须对这两者做出有效的反应 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的机制基础相关的研究。这款Mira的目标是 应用是为了解决我们对基因组稳定性途径及其如何形成的理解中的关键差距 在分裂和非分裂细胞中不同地使用，以适当地维持细胞内稳态。通过 细胞表面受体酪氨酸激酶(RTK)将外界信息从细胞外周传递到细胞核 通过PI3K-AKT信号反应生长因子，调节基因表达，从而促进 成长和/或生存。同样，DNA损伤会威胁到基因组的完整性，并在细胞核内被检测到 诱导DNA损伤反应(DDR)信号，以帮助DNA修复和细胞周期检查点。为了我们的研究 计划中，我们将解决与关键DDR因子如何促进细胞外生长相关的独特机制 分割和非分割信元中的信号串扰因素。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、 活细胞中的超分辨率显微镜和单分子跟踪。我们将检验这样一个假设，即 应被视为连接核质效应器的更广泛的应激反应网络，以维持 与生长因子信号通路交叉的生理动态平衡。如何实现这一目标 机械方面将是这一应用的主要焦点。第二个项目涉及到如何解决遗传毒性 细胞分裂中的压力影响DNA复制动力学并阐明新的调节分子 复制分叉恢复。基于我们的创新技术冈崎片段测序(OK-SEQ)，我们 能够直接量化在整个过程中在特定站点启动和终止复制分叉的效率 基因组。使用这项技术，我们将扩展我们的分析，以了解基因毒性侮辱和DNA 修复缺陷有助于使用全基因组分析来定位复制叉子介导的DNA断裂。 破译导致复制相关基因组不稳定性的机制可能会提供新的 癌症靶向治疗的途径。

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.