Replication of simple DNA repeats

描述(申请人提供)：简单DNA重复序列的扩展与人类近30种遗传性疾病有关。这项提议集中在导致重复扩张的分子机制上。在之前的资助期间，我们发现复制叉子在哺乳动物细胞中的可扩展重复序列中停滞不前。导致复制抑制的重复长度与其在人类家系中扩张的阈值长度非常接近。我们进一步研究了酵母中重复序列介导的复制阻断的控制，发现分叉稳定蛋白Tof1和MRc1通过可扩展的重复序列促进复制。最重要的是，我们开发了一种新的实验系统来分析酵母中的大规模重复扩张。这个系统的独特优势在于，它允许我们监测携带者大小的重复序列扩展到疾病大小范围。当复制分叉稳定剂Tof1失活时，扩张率显著升高，而在缺乏DNA解旋酶Sgs1或复制后修复调节因子Rad6的情况下，扩张率显著下降。总之，这些数据表明DNA复制和/或复制后修复与重复不稳定性有关；在这项提议中，我们将在酵母和哺乳动物细胞中进一步评估这一假说。我们将在我们的酵母实验系统中通过分析重复扩张率并可视化通过各种重复进行的复制分叉进程来研究各种三、四和五核苷酸重复序列的大规模扩展。扩展的重复序列抑制了酵母中的基因表达，就像它们在人类疾病中所做的那样。因此，我们将通过研究各种重复对转录、RNA剪接和RNA稳定性的影响来分析我们系统中基因抑制的机制。对于转录时不稳定的重复序列，我们将为其大规模扩展开发不同的系统，以便将它们定位在非转录区域。为了深入了解重复不稳定的遗传控制，我们将分析影响酵母DNA复制、修复和重组的大量突变体中的重复扩张和收缩。我们将进一步对酵母突变进行遗传筛选，这些突变显示使用突变的酵母基因组文库进行基因破坏时，大规模重复扩张率增加或重复收缩速度降低。在哺乳动物细胞中，我们将使用复制中间产物的二维电泳来评估pSV2neo Episome中各种可扩展重复序列的复制。我们还将研究可扩展重复序列是否会引发表观易损性。来自酵母筛选的哺乳动物同源基因的表达将被siRNAs敲除，以研究它们在重复介导的复制阻断和脆弱性中的作用。最后，我们将尝试开发一种新的系统，用于监测人类细胞的大规模重复扩增，使用专门设计的HyTK可选择盒结合Flip-in整合方法。这项提议的长期目标是了解导致人类反复扩张和收缩的分子机制。 与公共卫生相关：二十多种人类遗传性疾病是由人类基因中简单DNA重复的不可控扩张引起的。它们包括使人衰弱的神经疾病，如亨廷顿病、脆性X智力低下、强直性肌营养不良等。这一提议是为了揭开导致这一现象的分子机制。

DESCRIPTION (provided by applicant): Expansions of simple DNA repeats are implicated in nearly thirty hereditary disorders in humans. This proposal concentrates on molecular mechanisms responsible for repeat expansions. During the previously funded period we have found that replication forks stalled at expandable repeats in mammalian cells. The length of the repeat, which caused replication inhibition, closely matched the threshold length for its expansion in human pedigrees. We have further studied the control of repeat-mediated replication blockage in yeast to discover that the fork stabilizing proteins, Tof1 and Mrc1, facilitated replication through expandable repeats. Most significantly, we have developed a new experimental system to analyze large-scale repeat expansions in yeast. The unique advantage of this system is that it allowed us to monitor expansions of the carrier-size repeats well into the disease-size range. Expansion rates were strongly elevated upon inactivation of the replication fork stabilizer, Tof1, while significantly decreased in the lack of the DNA helicase Sgs1 or the post-replicative repair regulator Rad6. Altogether these data implicate DNA replication and/or post-replicative repair in repeat instability; we will further assess this hypothesis in yeast and mammalian cells in this proposal. We will study large-scale expansions of various tri-, tetra-and pentanucleotide repeats in our yeast experimental system by analyzing the rates of repeat expansions and visualizing the replication fork progression through various repeats. Expanded repeats inhibited gene expression in yeast as they do in human diseases. We will, therefore, analyze the mechanisms responsible for gene repression in our system by studying the effects of various repeats on transcription, RNA splicing and RNA stability. For repeats that are unstable when transcribed, we will develop a different system for their large-scale expansions, so that they are positioned in a non- transcribed area. To get insight into the genetic control of repeat instability, we will analyze repeat expansions and contractions in our large collection of mutants affecting DNA replication, repair and recombination in yeast. We will further perform genetic screens for yeast mutants that show either an increased rate of large-scale repeat expansions or a decreased rate of repeat contractions using gene disruption with a mutagenized yeast genomic library. In mammalian cells, we will evaluate replication of various expandable repeats in the pSV2neo episome using two-dimensional electrophoresis of replication intermediates. We will also study whether expandable repeats trigger episomal fragility. Expression of mammalian homologues of the genes, which came up from the yeast screens, will be knocked down by siRNAs to study their role in repeat-mediated replication blockage and fragility. Finally, we will attempt to develop a new system for monitoring large-scale repeat expansions in human cells using a specifically designed HyTK selectable cassette combined with the Flip-In integration approach. The long-term goal of this proposal is to understand molecular mechanisms responsible for repeat expansions and contractions in humans. PUBLIC HEALTH RELEVANCE: More than two dozen human hereditary diseases are caused by uncontrollable expansions of simple DNA repetitions within human genes. They include debilitating neurological disorders, such as Huntington's disease, fragile X mental retardation, myotonic dystrophy and others. This proposal is to unravel molecular mechanisms responsible for this phenomenon.