Mechanisms of Genomic Change in Budding Yeast

该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。基因组中的突变可能是由于DNA复制过程中的自然故障引起的，也可能是由辐射和某些化学物质等环境因素引起的;突变会促进癌症，衰老，神经退行性疾病和其他疾病，因此了解它们的起源和过程非常重要。一些频繁的“热点”突变出现在停滞的复制过程中，当新生的DNA链游荡并找到错误的链进行复制时。这个“模板切换”事件是这个项目的主要主题。目的是确定哪些基因影响这种突变事件的频率，以及哪些化学物质或其他环境因素可能促进它们。一个更广泛的影响目标是训练来自不同背景的学生如何进行科学研究，使用一种简单的生物体，芽殖酵母，可以很容易地在实验室中生长和遗传操作。将开发一个项目实验室，在一个支持和指导的环境中向没有研究实践和技术经验的学生介绍。 在DNA中不完全反向重复序列处的模板转换突变的遗传测定已经在酵母中工程化，并且将用于鉴定影响诱变的基因。初步证据表明，双链DNA断裂在修复过程中经常发生模板转换。HO内切核酸酶诱导的交配型转换系统将用于研究伴随双链断裂修复的DNA复制事件期间的模板转换。正常复制过程中的模板转换也将被检查以进行比较。最后，将使用模板转换报告系统筛选潜在诱变剂。研究结果将增加对诱变机制及其避免的认识，并提供与复制困难相关的DNA加工以及复制如何与修复协调的有价值的信息。该奖项反映了NSF的法定使命，并被认为值得通过使用基金会的智力价值和更广泛的影响审查标准进行评估来支持。

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.