Replication through DNA Structures and Consequences for Genome Stability

项目摘要/摘要 几乎一半的人类基因组由重复的DNA元件组成，约3%的 基因组由微卫星组成，即1-6个DNA碱基的短串联重复序列。多个重复序列 可以形成干扰复制和修复的替代DNA结构。这可能导致致病。 重复扩张，例如导致亨廷顿病的CAG/CTG扩张，强直性肌营养不良， 和许多脊髓小脑性共济失调。结构形成重复序列中的断裂会导致染色体缺失和 重排，这在经历复制压力的癌细胞中很常见。我的实验室的目标是 研究结构形成重复序列导致基因组不稳定的机制，并阐明细胞 为了防止这些有害的突变而进化的途径。我们最近发现，大片的 形成结构的CAG重复序列重新定位到核周，以促进通过纤维束和 防止染色体断裂和重复扩张。这一途径依赖于蛋白质在 通过总甲基化复制分叉，以及随后总甲基化蛋白与以下成分的相互作用 S期晚期核孔复合体，G2期释放回核内。 最近的数据显示，这一途径与几种类型的复制障碍有关，包括蛋白质 块和其他形成结构的重复序列。因此，更好地理解这样做的目的是至关重要的 迁往鼻咽癌及其在促进复制和防止基因组不稳定方面的作用，这是我们长期以来- 学期目标。 我们已经开发了一种系统来跟踪扩大的CAG管或其他结构的位置- 使用显微镜在细胞核中形成重复序列，辅之以生化技术来检测 与重复基因相互作用的蛋白质。我们将使用萌芽酵母(S.cerevisiae)系统，它允许我们 将这些方法与酵母系统强大的遗传学相结合。酵母复制与修复 途径与人类细胞保持高度保守，但较小的酵母基因组和 蛋白质组和细胞的野生型(未转化)状态是使我们能够显著 在我们的目标上取得进展。我们计划阐明将停滞不前的复制叉子重新安置到核设施的目的 孔复合体和那里发生的允许复制分叉重新启动的机制，使用已建立的 和新奇的方法。我们还将确定核孔连接的分叉重新启动如何阻止染色体 断裂和重复不稳定，并确定在叉子恢复期间如何发生重复扩展。我们的目标是 了解鼻咽癌依赖的复制体相关蛋白的修饰和发生在 复制障碍，并通过这样做了解维持基因组稳定的重要细胞过程。这是 这一点很重要，因为了解突变是如何产生的，对于制定预防其 发生了。

Project Summary/Abstract Almost half of the human genome is composed of repetitive DNA elements, and about three percent of the genome is composed of microsatellites, short tandem repeats of 1-6 DNA bases. Many repeat sequences can form alternative DNA structures that interfere with replication and repair. This can lead to disease-causing repeat expansions, such as the CAG/CTG expansions that cause Huntington’s disease, myotonic dystrophy, and many spinocerebellar ataxias. Breaks within structure-forming repeats cause chromosome deletions and rearrangements, which are common in cancer cells undergoing replication stress. The goal of my laboratory is to study mechanisms of genome instability caused by structure-forming repeats, and to elucidate cellular pathways that have evolved to prevent these deleterious mutations. We recently discovered that long tracts of structure-forming CAG repeats relocate to the nuclear periphery to facilitate replication through the tract and prevent chromosome breakage and repeat expansions. This pathway depends on modification of proteins at the replication fork by sumoylation, and subsequent interaction of the sumoylated proteins with components of the nuclear pore complex (NPC) in late S phase, followed by release back into the nuclear interior in G2 phase. Recent data shows that this pathway is relevant for several types of replication barriers, including protein blocks and other structure-forming repeats. Therefore, it is vital to better understand the purpose of this relocation to the NPC and its role in facilitating replication and preventing genome instability, which is our long- term goal. We have developed a system to follow the location of an expanded CAG tract or other structure- forming repeats in the cell nucleus using microscopy, complemented by biochemical techniques to detect proteins interacting with the repeat locus. We will use the budding yeast (S. cerevisiae) system which allows us to combine these approaches with the powerful genetics of the yeast system. Yeast replication and repair pathways retain a high level of conservation with human cells, but the smaller size of the yeast genome and proteome and wild-type (non-transformed) state of cells are advantages that will allow us to make significant progress on our goals. We plan to elucidate the purpose of relocation of stalled replication forks to the nuclear pore complex and the mechanisms that occur there to allow restart of replication forks, using both established and novel approaches. We will also determine how nuclear pore-linked fork restart prevents chromosome breaks and repeat instability and determine how repeat expansions occur during fork recovery. Our aim is to understand NPC-dependent modification of replisome-associated proteins and fork remodeling that occurs at replication barriers, and in so doing understand vital cellular processes that maintain genome stability. This is important because understanding how mutations arise is critical to developing strategies to prevent their occurrence.