Fidelity of Repair within Repetitive DNA

重复DNA在人类基因组中很常见。它容易发生长度变化、膨胀和收缩。这些变化导致基因组不稳定，可能导致疾病。亨廷顿氏病（HD）、脆性X综合征和肌萎缩侧索硬化症（ALS）是由重复扩增引起的疾病的例子。除了引起疾病的扩增，DNA重复序列是染色体脆性和重排的热点。癌细胞也表现出增加的脆弱性和染色体重排，因此更好地理解重复DNA内的修复保真度可能会导致对癌症病因的深入了解。我们已经建立了多个试验研究重复的不稳定性和脆弱性，使用酵母系统，这可以通过遗传操作。使用这些测定，我们已经表明，通过同源重组（HR）修复是CAG重复扩增的重要来源。酵母细胞含有一个扩大的重复序列，缺乏链退火蛋白Rad52，同源重组所需的，有高频率的染色体脆性，细胞周期停滞，细胞死亡，进一步暗示HR作为一个重要的过程中重复维护。此外，我们已经确定了一组有限的组蛋白修饰，控制扩大CAG重复内的修复保真度。细胞内有几种类型的HR，它们可以在不同的时间和空间位置发生。导致重复扩增的HR事件以及控制修复保真度的细胞过程目前尚不清楚。为了填补这些知识空白，我们建议与Jim Haber合作开发可控制的系统，以在重复DNA束内诱导HR，以确定哪些类型的HR修复产生重复扩增。此外，我们还将研究细胞核内的时间和位置如何影响修复途径的选择和保真度。最后，我们将研究组蛋白修饰如何在同源重组过程中控制修复保真度，并与谢尔盖·米尔金合作，筛选影响这一过程的其他因素。总体目标是确定细胞用于控制修复保真度和防止重复DNA内扩增的机制。

Repetitive DNA is common in the human genome. It is prone to length changes, expansions and contractions. These changes lead to genome instability that can cause disease. Huntington�s Disease (HD), Fragile X syndrome, and Amyotrophic Lateral Sclerosis (ALS) are examples of diseases caused by a repeat expansion. In addition to disease-causing expansions, DNA repeats are hotspots for chromosome fragility and rearrangements. Cancer cells exhibit increased fragility as well and chromosome rearrangements, thus a better understanding of repair fidelity within repetitive DNA could lead to insights into cancer etiology. We have established multiple assays for studying repeat instability and fragility using a yeast system, which can be manipulated genetically. Using these assays, we have shown that repair via homologous recombination (HR) is a significant source of CAG repeat expansions. Yeast cells containing an expanded repeat tract and lacking the strand annealing protein Rad52, required for homologous recombination, have high frequencies of chromosome fragility, cell cycle arrest, and cell death, further implicating HR as an important process in repeat maintenance. Moreover, we have identified a limited set of histone modifications that control the fidelity of repair within an expanded CAG repeat. There are several types of HR within cells, which can occur with different temporal and spatial locations. The HR event which is causing repeat expansions, and the cellular processes that control repair fidelity, are currently unclear. To fill these gaps in knowledge, we propose to develop, in collaboration with Jim Haber, controllable systems to induce HR within a repetitive DNA tract, in order to determine which types of HR repair generate repeat expansions. In addition, we will investigate how timing and location within the nucleus influence repair pathway choice and fidelity. Lastly, we will investigate how histone modifications control repair fidelity during homologous recombination, and, in collaboration with Sergei Mirkin, screen for additional factors that influence this process. The overall goal is to determine the mechanisms the cell uses to control repair fidelity and prevent expansions within repetitive DNA.