Replication of Simple DNA Repeats

描述(申请人提供)：二十多种人类遗传性疾病是由不同基因中简单DNA重复序列的不可控扩展引起的。本研究旨在研究体内可扩增的DNA重复序列的复制以及DNA复制在其扩增中的作用。在之前的资助期间，我们开发了一种新的策略来克隆酵母中的长不间断重复序列，并使用复制中间产物的电泳分析来研究它们的复制。我们已经发现，通过可扩展的三核苷酸重复进行的复制分叉的进程受到了损害。我们进一步观察到，复制装置中影响滞后链DNA合成的突变可以增加重复扩张的速度。我们还发现，重复序列的复制及其扩展的倾向可以取决于它们相对于复制起始点的方向。我们的工作假设是，复制在重复运行时停滞是主要事件，这可能导致复制分叉试图逃离停滞位置时重复的不稳定。在这项建议中，我们将在酵母和哺乳动物系统中评估这一假说。我们将继续研究可扩展重复序列的复制，包括那些不同于三联体重复序列的复制，以确认复制停滞是这类DNA序列的普遍现象。我们将进一步开发一个实验系统，使我们能够将复制停滞与酵母染色体环境中各种DNA重复的大规模扩张联系起来。我们将利用这一系统来揭示顺式作用因子在重复扩增中的作用，例如复制起点。我们最终将把这些研究扩展到哺乳动物系统。我们将通过各种方法分析携带可扩展重复序列的附着体在培养的哺乳动物细胞中的复制。我们将进一步研究可扩展重复序列的长度多态性和脆性，这些重复序列在哺乳动物的表观染色体中复制。我们的长期目标是了解人类重复扩张的机制。

DESCRIPTION (provided by applicant): More than two dozens of human hereditary disorders are caused by uncontrollable expansions of simple DNA repeats within various genes. This proposal is to study replication of expandable DMA repeats in vivo and the role of DNA replication in their expansions. During the previously funded period, we have developed a novel strategy for cloning of long non-interrupted repeats in yeast and studied their replication, using electrophoretic analysis of replication intermediates. We have discovered that progression of the replication fork through expandable trinucleotide repeats is compromised. We have further observed that mutations in the replication apparatus, that affect the lagging strand DNA synthesis, can increase the rate of repeat expansions. We have also found that repeats' replication and their propensity to expand can depend on their orientation relative to replication origins. Our working hypothesis is that replication stalling at a repetitive run is the primary event, which could result in the repeat's instability during the replication fork attempts to escape from the stall site. In this proposal, we will assess this hypothesis in both yeast and mammalian systems. We will continue studying replication of expandable repeats, including those, differing from triplet repeats, to affirm that replication stalling is the universal phenomenon for this class of DNA sequences. We will further develop an experimental system, which would allow us to correlate replication stalling with large-scale expansions for various DNA repeats in yeast chromosomal environment. We will exploit this system to unravel the role of cis-acting factors, such as replication origins, in repeat expansions. We will finally extend these studies into mammalian system. We will analyze replication of episomes, which carry expandable repeats, in cultured mammalian cells by various approaches. We will further investigate the length polymorphism and fragility of expandable repeats, replicating in mammalian episomes. Our long-term goal is to understand the mechanisms of repeat expansions in humans.