Replication of Simple DNA Repeats

描述（申请人提供）：超过20种人类遗传性疾病是由各种基因内简单DNA重复序列的不可控制的扩增引起的。本研究旨在研究DNA可扩展重复序列在体内的复制以及DNA复制在其扩展中的作用。在以前的资助期间，我们已经开发了一种新的策略克隆长的非中断重复在酵母和研究它们的复制，使用复制中间体的电泳分析。我们已经发现，通过可扩展的三核苷酸重复的复制叉的进展受到损害。我们进一步观察到，影响滞后链DNA合成的复制装置中的突变可以增加重复扩增的速率。我们还发现重复序列的复制和它们的扩展倾向可以取决于它们相对于复制起点的方向。我们的工作假设是，复制停止在重复运行是主要事件，这可能会导致重复的不稳定性，在复制叉尝试逃离失速站点。在这个提议中，我们将在酵母和哺乳动物系统中评估这一假设。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重复序列的重复序列，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，这将使我们能够在酵母染色体环境中将复制停滞与各种DNA重复序列的大规模扩增相关联。我们将利用这个系统来解开顺式作用因子，如复制起点，在重复扩增的作用。我们最终将把这些研究扩展到哺乳动物系统。我们将分析复制的附加体，携带可扩展的重复序列，在培养的哺乳动物细胞通过各种方法。我们将进一步研究可扩展重复序列的长度多态性和脆性，在哺乳动物游离体中复制。我们的长期目标是了解人类重复扩增的机制。

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.