Replication of Simple DNA Repeats

摘要 简单DNA重复序列的扩增与30多种遗传性神经系统和 人类的发育障碍这一建议致力于分子机制负责 重复扩张。我的实验室是第一个证明可扩展的重复序列会阻碍复制叉的进展的实验室。 在体内的长度依赖性的方式，让人想起他们在人类的不稳定模式。这种现象 在从细菌到哺乳动物细胞研究的所有实验系统中观察到，随后通过 许多其他实验室。这使我们和其他人制定了重复扩张的复制模型，规定 当复制叉试图逃离“重复陷阱”时，可以添加重复。期间 在以前的资助期间，我们已经开发出了第一种系统来检测大规模的重复扩张， 酵母、S.啤酒。重要的是，在这个系统中看到的重复扩增的许多特征与 那些在人类谱系中观察到的。此外，全基因组水平的遗传筛查显示， 重复扩增主要发生在其复制过程中，可能涉及DNA模板 切换我们还发现，虽然转录不是重复扩增所必需的，但似乎 大力推动这一进程。我们计划继续这些重复的分子机制的研究， 在酵母和哺乳动物细胞中的扩增。 我们将研究转录和转录-复制相互作用在扩增过程中的作用， 开发实验系统，其中重复的转录是已知的，并且可以控制。这些 新的实验系统使我们能够进行并排比较的膨胀机制， 不同的DNA重复序列我们目前的假设是每个DNA的扩张途径 重复的能力取决于它形成DNA发夹的倾向与它形成DNA发夹的倾向之间的微妙平衡。 偏离复制叉进程。这一想法将通过比较膨胀率和规模来确定 在相同的实验环境中进行不同的重复，以及进行比较遗传分析， 通过候选基因的方法进行不同的重复扩增。我们还将寻找重复序列的遗传修饰剂， 通过进行无偏诱变筛选，然后进行全基因组测序， 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， 在SUP 4-o抑制tRNA等位基因的内含子中含有疾病大小的重复序列的可选择盒。我们 然后，将通过用酵母迷你筛选来识别影响收缩过程的基因， 转座子基因组文库。最后，我们将继续致力于一个可选择的系统，用于分析重复 在培养的哺乳动物细胞中不稳定。它是基于一个盒子的整合，其中携带HyTK 在FMR 1启动子的控制下，在其5 'UTR中具有载体长度（CGG）n重复序列的基因，转化为独特的 在一些实施方案中，所述方法包括在鼠红系白血病或人HEK-293细胞的基因组位点上进行修饰。我们将使用这种方法来检测 重复扩增和重复介导的诱变，并解开负责的机制， 使用siRNA或shRNA针对在我们的酵母筛选中鉴定的候选基因的不稳定性。 这项建议的长期目标是了解负责重复的分子机制。 人类的不稳定性。从长远来看，我们希望了解这些机制的细节，足以提出 用于治疗这些使人衰弱的人类疾病的新的治疗策略。

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.