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、扩张和收缩。这些变化会导致基因组不稳定，从而引发疾病。亨廷顿氏病（HD），脆性X综合征和肌萎缩侧索硬化症（ALS）是由重复扩张引起的疾病的例子。除了引起疾病的扩展外，DNA重复序列是染色体脆弱和重排的热点。癌细胞也表现出增加的脆弱性和染色体重排，因此更好地了解重复DNA内的修复保真度可能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，这可以操纵基因。通过这些实验，我们发现同源重组（HR）修复是CAG重复扩增的重要来源。酵母细胞含有扩增的重复束，缺乏同源重组所需的链退火蛋白Rad52，染色体脆性、细胞周期阻滞和细胞死亡的频率很高，进一步表明HR是重复维持的重要过程。此外，我们已经确定了一组有限的组蛋白修饰，这些修饰控制着扩展CAG重复序列中修复的保真度。细胞内有几种类型的HR，可能发生在不同的时间和空间位置。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合作开发可控系统，在重复DNA束中诱导HR，以确定哪种类型的HR修复产生重复扩增。此外，我们将研究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控制同源重组过程中的修复保真度，并与Sergei Mirkin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来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.