Role of ISWI Chromatin Remodeler in Transcription-coupled Repair and Genome Stability

智力上的功绩。每个有机体的细胞都必须保存基因组，以防止突变。重复DNA在基因组中很常见，对细胞来说是一个特别的挑战，因为在复制或修复过程中可能会发生滑移。特别是，结构形成重复序列，如CAG/CTG三联体重复序列，已被证明特别容易在拷贝数上增加和减少。了解重复DNA中的突变过程是至关重要的，特别是当突变发生在可能影响基因功能的区域时。转录区域内使用的关键修复系统之一是转录偶联修复。最近的发现表明，在转录偶联修复过程中，需要一种新的因子Isw1染色质重塑蛋白来防止CAG的扩张。该项目的具体目标是确定Isw1在转录偶联修复中的作用，以及它如何控制CAG重复内的修复保真度以防止扩张。模型真核生物酿酒酵母(面包酵母)将被使用，因为它表现出类似于人类细胞的重复不稳定，但提供了强大的遗传和分子工具，将被用来解决所提出的问题。这项研究将为包括酵母和人类在内的真核生物的突变过程提供基本的见解。此外，转录偶联修复中一种新的参与者Isw1蛋白的发现，为更好地理解这一重要的DNA修复途径以及它如何在染色质的背景下工作创造了机会，染色质是DNA在细胞内包装时采取的形式。Isw1可以“重塑”染色质，或改变其结构以便于获取DNA。因此，该项目的结果也有很高的潜力，可以显着提高目前对染色质重塑在DNA修复中的作用的有限认识。除了创造新的科学知识外，该项目还将对本科生的教育产生更广泛的影响。传统上，在本科课程中教授的实验室练习包括让学生遵循既定的规程并牢记特定结果的活动。然而，真正的科学很少遵循这种可预测的模式。该项目的一部分将是利用我们的模型系统的多功能性来开发一门新的实验室课程，旨在让塔夫茨大学的本科生参与分子遗传学领域的新研究。这门新课程将允许本科生体验“非食谱”的实验室研究，并通过对防止突变和基因组重排的细胞因子进行开放式筛选来做出新的发现。目前只有一小部分学生有这个机会，因此这个实验室的提供将极大地扩大从事重大科学研究和批判性思维的学生的数量。从事转录偶联修复项目的年轻科学家也将参与新课程的设计和实施，使他们有机会将他们的研究努力与课程设计和教学培训结合起来。为了让其他教育工作者使用这些教学模块，一旦定稿，课程材料、方案和试剂将通过出版提供给更大的遗传学社区。

Intellectual Merit. Cells of every organism must preserve the genome to prevent mutations. Repetitive DNA is common in genomes and presents a particular challenge for cells, as slippage can occur during replication or repair. In particular, structure-forming repeats, such as CAG/CTG triplet repeats, have been shown to be particularly prone to gains and losses in copy number. It is essential to understand mutational processes within repetitive DNA, especially when mutations occur in a region that could affect gene function. One of the key repair systems used within transcribed regions is transcription-coupled repair. Recent discovery indicates that a new factor, the Isw1 chromatin remodeler protein, is needed to prevent CAG expansions during transcription-coupled repair. The specific objective of this project is to determine the role of Isw1 in transcription-coupled repair, and how it controls fidelity of repair within a CAG repeat to prevent expansions. The model eukaryote Saccharomyces cerevisiae (baker's yeast) will be used since it exhibits repeat instability similar to human cells, yet offers powerful genetic and molecular tools that will be employed to address the proposed questions. This research will provide fundamental insight into mutational processes in eukaryotic organisms, including yeast and humans. In addition, the discovery of a new player in transcription-coupled repair, the Isw1 protein, creates an opportunity to better understand this important DNA repair pathway and how it operates in the context of chromatin, which is the form that DNA takes when packaged within cells. Isw1 can "remodel" chromatin, or change its structure to facilitate access to the DNA. Thus the results of this project also have a high potential to significantly advance the current limited knowledge of the role of chromatin remodeling in DNA repair.Broader Impacts. In addition to creating new scientific knowledge, this project will have a broader impact on education of students at the undergraduate level. Traditionally laboratory exercises taught in undergraduate courses consist of activities where students follow a set protocol with a particular result in mind. However, real science rarely follows this predictable pattern. Part of this project will be to take advantage of the versatility of our model system to develop a new laboratory course designed to engage Tufts undergraduates in novel research in the area of molecular genetics. This new course will allow undergraduates to experience "non-cookbook" laboratory research and make new discoveries by doing an open-ended screen for cellular factors that protect against mutations and genome rearrangements. Currently this opportunity is available to only a small number of students, so the availability of this laboratory will significantly broaden the number of students engaged in significant scientific research and critical thinking. The young scientists working on the transcription-coupled repair project will also be involved in the design and implementation of the new course, giving them the opportunity to combine their research efforts with course design and training in teaching. To allow other educators to utilize these teaching modules, once finalized, the course materials, protocols and reagents will be made available to the larger Genetics community through publication.