Mechanisms of replication stress-induced chromosome fragility

项目总结/摘要 在这项研究中，我们的目标是解决DNA复制应激如何导致 染色体脆性，这是基因组不稳定性和癌症的潜在机制之一 发展染色体脆性位点是诱导DNA双链断裂的有效首选位点 在复制压力下，是我们基因组的内在和脆弱的特征。然而， 由于缺乏合适的基因组规模检测，这些断裂位点中的大多数仍然未知 方法.此外，这也是一个长期存在的问题，为什么不同的复制抑制剂以及不同的 细胞类型产生不同的断裂形成谱。我们假设复制抑制剂 同时使复制叉不稳定并引发独特的基因表达模式，从而引起 复制和转录之间的非计划性冲突，最终导致DNA链断裂。我们建议 通过在酵母模型和人类细胞系中使用方法的组合来测试这一假设。我们将 首先利用遗传上可接受的酵母模型来直接测试我们的核心假设。我们将学习 复制和转录动力学之间的相互作用如何影响DSB形成和基因组稳定性。 然后，我们将系统地绘制酵母和人类基因组中药物特异性CFS的形成。这些 实验将能够识别与DSB和潜在疾病相关的基因组特征， 相关基因，如肿瘤抑制基因。我们还将使用已建立的重组系统， 酵母模型来测试与DNA断裂相关的基因组特征的功能。我们的研究将填补 我们对染色体脆性位点位置的认识上的差距，使我们能够发现新的癌症- 相关基因，并提供深入了解基因组不稳定性的机制。 拟议的项目包括分子生物学，酵母遗传学，哺乳动物细胞培养， 基因组学和生物信息学。冯文毅博士领导的研究团队包括一名博士前实习生 （第二年在博士学位。计划）和两到三名本科生（通过夏季研究 奖学金计划提供的上州医科大学和学分一到两个学期的研究 课程由锡拉丘兹大学提供），一个研究支持专家，和一个未来的招聘 博士后实习生。博士前实习生是在入门级酵母遗传学和哺乳动物细胞培养 操作，预计将在本项目期间完成培训。培训 一般只有最低限度实验技能的本科生，预期每个受训者能持续一到两年。 最后，拟招聘的博士后研究生预计具有哺乳动物细胞培养经验 和/或下一代测序，预计也将在本项目期间完成培训。

PROJECT SUMMARY/ABSTRACT In this research proposal we aim to address the question how DNA replication stress causes chromosome fragility, which is one of the underlying mechanisms of genome instability and cancer development. Chromosome fragile sites are effectively preferred sites of DNA double strand breaks inducible upon replication stress, and are an intrinsic and vulnerable feature of our genome. Yet, the locations of the majority of these break sites still remain unknown due to the shortage of suitable genome scale detection methods. Moreover, it is also a long-standing question why different replication inhibitors as well as different cell types produce distinct spectra of breakage formation. We hypothesize that replication inhibitors simultaneously destabilize replication forks and elicit unique patterns of gene expression, thus causing unscheduled clash between replication and transcription and ultimately DNA strand breaks. We propose to test this hypothesis by using a combination of approaches in a yeast model and in human cell lines. We will first take advantage of the genetically amenable yeast model to directly test our core hypothesis. We will learn how the interplay between replication and transcription dynamics impacts DSB formation and genome stability. We will then systematically map drug-specific CFS formation in both yeast and human genomes. These experiments will enable the identification of genomic features associated with DSBs and potentially disease- associated genes such as tumor suppressors. We will also use an established recombination system in the yeast model to test the functionality of the genomic features associated with DNA breaks. Our research will fill the gap in our knowledge of the locations of chromosome fragile sites, enable the discovery of new cancer- associated genes and provide insights into the mechanisms of genome instability. The proposed project encompasses molecular biology, yeast genetics, mammalian cell culture, genomics and bioinformatics. The research team led by Dr. Wenyi Feng includes one predoctoral trainee (second year in a Ph.D. program) and two to three undergraduate trainees (through the summer research fellowship program provided by Upstate Medical University and a credited one to two semester research course provided by Syracuse University), one research support specialist, and one future recruit of a postdoctoral trainee. The predoctoral trainee is at entry level to yeast genetics and mammalian cell culture manipulations and is expected to complete training within the duration of this project. Training for the undergraduates who typically have minimal experimental skills is expected to last one to two years per trainee. Finally, the postdoctoral trainee to be recruited is expected to have experience in mammalian cell culture and/or next-generation sequencing and is also expected to complete training within the duration of this project.