Fork Restart at Replication Barriers and Effects on Genome Stability

项目总结 基因组的复制是生命所必需的基本过程。在复制期间 组成基因组的数百万个DNA碱基对，复制叉遇到了障碍 必须克服的问题，包括DNA结构、强结合蛋白或受损或 交联碱基。在解决或绕过障碍后，复制分叉必须重新启动，因为 尽可能准确地避免染色体断裂；这是发生这种情况的一种机制 涉及通常用于同源重组过程的蛋白质，这一过程 通过DNA链入侵而发生。最近，人们已经认识到，这个过程可能是 由核位置调节，停滞的复制叉重新定位到核外围 重启重新开始。本应用程序的长期目标是了解 分叉重新启动，它的核位置的调节，以及它在防止基因组不稳定和 有害的染色体断裂。其具体目的是：(1)阐明核孔的作用 在分叉重新启动中的定位和重组的调节，以及(2)研究 叉子在重复不稳定时重新启动。我们已经证明了扩展的CAG重复序列，它 形式DNA结构，特别依赖分叉重新启动来防止拷贝数更改和 染色体的脆弱性。因此，研究结构形成过程中的再启动问题就显得尤为重要 重复，因为它们是基因组中突变的热点，它们的扩张可能会导致 退行性疾病，如亨廷顿病、强直性肌营养不良和肌萎缩侧索硬化症。此外, 结构形成重复序列是一种脆弱的位点，可以破坏并引发重要DNA的缺失 在癌症启动的早期步骤中的序列，复制应激加剧了癌症的启动 发生在癌细胞中。这项研究的结果将是更好地理解细胞 用于应对复制压力的机制，以及这些机制如何出错 导致基因突变。这些信息应该为如何支持或干预提供洞察力 通过这些过程，要么防止突变，要么针对突变细胞进行消除。

PROJECT SUMMARY Replication of the genome is a fundamental process necessary for life. During the replication of the millions of DNA base-pairs that make up a genome, the replication fork encounters barriers that must be overcome, including DNA structures, strongly bound proteins, or damaged or cross-linked bases. After the barrier is resolved or bypassed, the replication fork must restart as accurately as possible to avoid chromosome breakage; one mechanism by which this happens involves proteins that are normally used for a process called homologous recombination, which occurs by DNA strand invasion. Recently, it has been recognized that this process may be regulated by nuclear location, with the stalled replication fork relocating to the nuclear periphery to undergo restart. The long-term objective of this application is to understand the process of fork restart, its regulation by nuclear location, and its role in preventing genome instability and deleterious chromosome breaks. The specific aims are (1) to elucidate the role of nuclear pore localization in fork restart and regulation of recombination, and (2) to investigate the effect of fork restart on repeat instability. We have shown that expanded CAG repeat sequences, which form DNA structures, are particularly reliant on fork restart to prevent copy number changes and chromosome fragility. Thus it is of critical importance to study restart at structure-forming repeats as they are hotspots for mutation in the genome, and their expansion can cause degenerative diseases such as Huntington's disease, myotonic dystrophy, and ALS. In addition, structure-forming repeats are fragile sites that can break and provoke deletion of vital DNA sequences during early steps of cancer initiation, which is exacerbated by replication stress occurring in cancer cells. The outcome of this research will be a better understanding of cellular mechanisms used to cope with replication stress, and how these mechanisms can go wrong to cause genetic mutation. This information should provide insights into how to bolster or interfere with these processes, to either prevent mutation or target mutated cells for elimination.