The Interplay of Replication and Transcription in Fragility of Structured DNA

DNA的完整性是生命的基础，但有时DNA可能会断裂，导致遗传物质丢失，甚至细胞死亡。有趣的是，染色体的某些区域，也就是所谓的脆性部位，往往更容易断裂。这些脆弱的部位是DNA突变的常见来源，例如遗传物质的丢失或获得。因此，了解染色体脆弱性的基础将有助于深入了解疾病的原因和基因组进化。这一课题引起了学习分子生物学和遗传学的大学生的极大兴趣，并开发了一个分子遗传学项目实验室，每年向16名学生提供研究经验，使用酵母模型系统调查染色体脆性的原因。本课程的目标是确定防止脆弱性的新途径，并给学生提供真实的研究体验。这个项目将支持实验室课程以及本科生和研究生的后续实验，以获得关于防止有害突变的重要途径的新知识。人类基因组中的许多脆弱部位既与干扰复制的DNA结构有关，也与转录区域有关。人们怀疑，形成结构的DNA序列、转录和复制的共同出现可能会导致这些区域易于脆弱，然而它们之间的相互作用却鲜为人知。在酿酒酵母中已经开发了遗传系统来测量在特定结构处诱导的染色体断裂-形成导致DNA脆弱性的重复DNA序列。在这个系统中，可以通过DNA结构控制转录状态和复制时间，以直接测量DNA断裂的后果。蛋白质在引起或保护DNA断裂方面的作用将被确定，包括已识别的核酸酶MLH1-MLH3和MUS81的作用。这些工具将被用来阐明转录和复制在导致两个生理上相关的脆性序列的脆弱性方面的贡献和相互作用：(1)扩大的CAG重复序列和(2)来自人类脆性位点FRA16D的长AT重复序列。所获得的知识将加强对脆弱性和由此导致的基因组不稳定导致染色体删除和重新排列的机制的理解。这一奖项反映了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 integrity of DNA is fundamental to life, but sometimes DNA can break, leading to loss of genetic material or even cell death. Interestingly, certain areas of chromosomes, known as fragile sites, tend to break more frequently. These fragile sites are a frequent source of DNA mutations, for example loss or gain of genetic material. Thus, an understanding of the basis of chromosome fragility will provide insight into causes of disease and of genome evolution. This topic is of great interest to college students studying molecular biology and genetics, and a Molecular Genetics Project Laboratory was developed to provide a research experience to 16 students a year, investigating the causes of chromosome fragility using a yeast model system. The goal of the course is to identify new pathways that protect against fragility, and give the students an authentic research experience. This project will support both the laboratory class as well as follow-up experiments by undergraduate and graduate students, to gain new knowledge about important pathways that prevent deleterious mutations. Many fragile sites in the human genome are associated with both DNA structures that interfere with replication and transcribed regions. It is suspected that the co-incidence of structure-forming DNA sequences, transcription, and replication may combine to cause these regions to be prone to fragility, however the interplay between them is poorly understood. Genetic systems have been developed in the yeast Saccharomyces cerevisiae to measure chromosome breaks that are induced at specific structure-forming repetitive DNA sequences that cause DNA fragility. In this system, transcription state and replication timing through the DNA structure can be controlled, to directly measure the consequences for DNA breakage. The role of proteins in causing or protecting against DNA breaks will be determined, including the action of identified nucleases Mlh1-Mlh3 and Mus81. These tools will be employed to elucidate the contributions and interplay of transcription and replication in causing fragility at two physiologically relevant fragile sequences: (1) expanded CAG repeats and (2) a long AT repeat from the human fragile site FRA16D. The knowledge gained will enhance understanding of mechanisms of fragility and the resulting genome instability that leads to chromosomal deletions and rearrangements.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.