Mechanisms of Genomic Change in Budding Yeast

这个项目的总体目标是研究细胞如何保持其基因组DNA的稳定性。一些频繁的“热点”突变出现在停滞的复制过程中，当时新生的DNA链四处游荡，找到了要复制的错误链。这一“模板切换”事件是本项目的主要主题。其目的是确定哪些基因会影响此类突变事件的频率，以及哪些化学物质或其他环境因素可能会促进它们的发生。一个更广泛的影响目标是，培训来自不同背景的学生如何使用一种简单的有机体--发芽酵母--进行科学研究，这种生物可以很容易地在实验室中培养和进行基因操作。将开发一个项目实验室，在一个支持性和指导性的环境中，向没有研究实践和技术经验的学生介绍。对DNA中不完美反向重复序列中模板切换突变的遗传分析已经在酵母中进行了改造，并将用于识别影响突变的基因。初步证据表明，双链DNA断裂在修复过程中经常发生模板切换。HO内切酶诱导的交配型切换系统将用于研究伴随双链断裂修复的DNA复制事件中的模板切换。还将检查正常复制过程中的模板切换，以进行比较。最后，使用模板切换报告系统筛选潜在的诱变剂。这一结果将增加对突变及其避免机制的了解，并提供与复制困难相关的DNA处理以及复制与修复如何协调的有价值的信息。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

The overall goal of this project is to study how cells maintain the stability of their genomic DNA. Mutations can arise in genomes due to natural failures during DNA replication or can be induced by environmental factors such as radiation and certain chemicals; mutations promote cancer, aging, neurodegenerative and other diseases, hence it is important to understand their genesis and processing. Some frequent “hotspot” mutations arise during stalled replication, when the nascent DNA strand wanders and finds a wrong strand to copy. This “template-switch” event is the main subject of this project. The aims are to determine what genes influence the frequency of such mutation events and what chemicals or other environmental factors might promote them. A broader impact goal is to train students from diverse backgrounds on how to conduct scientific research, using a simple organism, budding yeast, that can be easily grown and genetically manipulated in the laboratory. A project lab will be developed to introduce students with no prior experience to research practice and techniques, in a supportive and mentored environment. Genetic assays for template-switch mutations at imperfect inverted repeats in DNA have been engineered in yeast and will be used to identify genes that influence mutagenesis. Preliminary evidence suggests that double-strand DNA breaks frequently undergo template-switching during repair. The HO endonuclease-induced mating-type switching system will be used to study template-switching during DNA replication events that accompany double-strand break repair. Template-switching during normal replication will also be examined for comparison. Finally, potential mutagens will be screened using the template-switch reporter system. The outcomes will increase knowledge about the mechanisms of mutagenesis and its avoidance, and provide valuable information about DNA processing associated with replication difficulties and how replication is coordinated with repair.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.