Replication of Simple DNA Repeats

摘要 简单DNA重复序列的扩展与30多种遗传性神经和 人类的发育障碍。这项提议致力于研究导致分子机制 重复扩展。我的实验室是第一个证明可扩展重复阻碍复制分叉进展的实验室 活体以长度依赖的方式存在，这让人想起它们在人类中的不稳定模式。这一现象是 在从细菌到哺乳动物细胞的所有实验系统中观察到，随后证实了 很多其他的实验室。这导致我们和其他人制定了重复扩展的复制模型，规定 当复制分叉试图逃离“重复陷阱”时，可以添加该重复。在.期间 在之前的资助期间，我们开发了第一个系统来检测大规模重复扩张 酵母，酿酒酵母。重要的是，在这个系统中看到的重复扩展的许多特征与之密切匹配 在人类谱系中观察到的那些。此外，全基因组水平的遗传筛查显示， 重复扩增主要发生在它们的复制过程中，可能涉及DNA模板 正在切换。我们还发现，虽然转录对于重复扩增不是必需的，但它似乎是必要的 为这一进程作出强有力的贡献。我们计划继续对重复序列的分子机制进行研究 在酵母和哺乳动物细胞中的扩张。 我们将通过以下方式研究转录和转录-复制相互作用在扩张过程中的作用 开发实验系统，其中重复的转录是已知的并可以控制的。这些 新的实验系统使我们能够对扩展机构进行并排比较 各种各样的DNA重复。我们目前的工作假设是每个个体DNA的扩展路径 重复依赖于其形成DNA发夹的倾向与其形成DNA发夹的倾向之间的微妙平衡 偏离复制分叉进程。这一想法将通过比较扩张速度和规模来确定 在相同的实验环境中对不同的重复序列进行分析，以及进行比较遗传分析 通过候选基因方法进行不同重复序列的扩增。我们还将寻找重复序列的遗传修饰物 通过进行无偏向诱变筛选和全基因组测序来鉴定 致病突变。为了研究酵母中的大规模重复收缩，我们将开发一个系统，利用 在SUP4-o抑制因子tRNA等位基因的内含子中包含疾病大小重复的可选盒。我们 然后将通过使用酵母迷你进行筛选来识别影响收缩过程的基因 转座子基因组文库。最后，我们将继续研究用于重复分析的可选系统 培养的哺乳动物细胞的不稳定性。它是基于集成了一个带有HyTK的盒式磁带 基因在FMR1启动子的控制下，在其5‘端重复(CGG)n个重复序列，形成一个独特的 小鼠红系白血病细胞或人HEK-293细胞的基因组位置。我们将使用这种方法来检测 重复扩增和重复介导的突变，并揭开负责 使用siRNA或shRNA对我们的酵母筛选中确定的候选基因进行不稳定。 这项提议的长期目标是了解导致重复的分子机制。 人类的不稳定性。从长远来看，我们希望对这些机制有足够的了解，以便提出 治疗这些令人衰弱的人类疾病的新治疗策略。

ABSTRACT Expansions of simple DNA repeats are implicated in more than thirty hereditary neurological and developmental disorders in humans. This proposal is devoted to the molecular mechanisms responsible for repeat expansions. My lab was the first to show that expandable repeats stall replication fork progression in vivo in a length-dependent manner, reminiscent of their instability pattern in humans. This phenomenon was observed in all experimental system studied from bacteria to mammalian cells and subsequently confirmed by many other labs. This led us and others to formulate the replication model for repeat expansions stipulating that repeats could be added as the replication fork attempts to escape from the "repetitive trap". During the previously funded period, we have developed first-of-a-kind system to detect large-scale repeat expansions in yeast, S. cerevisiae. Importantly, many features of repeat expansions seen in this system closely matched those observed in human pedigrees. Furthermore, genetic screening at the whole-genome level revealed that repeat expansions occur primarily during their replication in the process likely involving DNA template switching. We also found that while transcription is not necessary for the repeat expansions, it seems to strongly contribute to the process. We plan to continue these studies of the molecular mechanisms of repeat expansions in yeast and mammalian cells. We will study the role of transcription and transcription-replication interplay in the expansion process by developing experimental systems, in which transcription of the repeat is known and can be controlled. These new experimental systems enable us to carry out side-by-side comparisons of the expansion mechanisms for various DNA repeats. Our current working hypothesis is that expansion pathway for each individual DNA repeat can depend on a delicate balance between its propensity to form DNA hairpins versus its propensity to deviate the replication fork progression. This idea will be ascertained by comparing expansion rates and scales for different repeats in the same experimental settings, as well as by carrying out comparative genetic analysis of different repeat expansions via candidate gene approach. We will also search for genetic modifiers of repeat expansions by conducting an unbiased mutagenic screen followed by whole-genome sequencing to identify causative mutations. To study large-scale repeat contractions in yeast, we will develop a system utilizing a selectable cassette that contains disease-sized repeats in the intron of the SUP4-o suppressor tRNA allele. We will then identify genes affecting the contraction process by carrying out a screen with the yeast mini- transposon genomic library. Finally, we will continue working on a selectable system for the analysis of repeat instability in cultured mammalian cells. It is based on the integration of a cassette, which carries the HyTK gene under the control of the FMR1 promoter with carrier-length (CGG)n repeats in its 5'UTR, into a unique genomic site of the murine erythroid leukemia or human HEK-293 cells. We will used this approach to detect repeat expansions and repeat-mediated mutagenesis, and to unravel the mechanisms responsible of the instability using siRNA or shRNA against the candidate genes identified in our yeast screens. The long-term goal of this proposal is to understand the molecular mechanisms responsible for repeat instability in humans. In the long run, we hope to understand these mechanisms in details sufficient to propose new therapeutic strategies for treatments of these debilitating human diseases.