Replication through DNA Structures and Consequences for Genome Stability

通过 DNA 结构进行复制以及对基因组稳定性的影响

• 批准号: 10544323
• 负责人: CATHERINE H FREUDENREICH
• 金额: $ 39万
• 依托单位: TUFTS UNIVERSITY MEDFORD
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-01-01 至 2026-12-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10544323
• 关键词: Back; Biochemical; CAG repeat; Cell Nucleus; Cell physiology; Cells; Chromosomal Breaks; Chromosome Breakage; Chromosome Deletion; Complement; DNA; DNA Structure; DNA replication fork; Data; Disease; Elements; Failure; G2 Phase; Genetic; Genome; Genome Stability; Genomic Instability; Goals; Human; Human Genome; Huntington Disease; Laboratories; Link; Location; Microsatellite Repeats; Microscopy; Modification; Mutation; Myotonic Dystrophy; Nuclear; Nuclear Pore; Nuclear Pore Complex; Pathway interactions; Post-Translational Protein Processing; Proteins; Proteome; Recovery; Replication-Associated Process; Role; S phase; Saccharomyces cerevisiae; Saccharomycetales; Short Tandem Repeat; Spinocerebellar Ataxias; Structure; Sumoylation Pathway; System; Techniques; Yeasts; base; cancer cell; cancer initiation; chromosome mutation; disease-causing mutation; experimental study; novel strategies; prevent; repaired; replication stress; yeast genetics; yeast genome

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.

项目总结/摘要 几乎一半的人类基因组由重复的DNA元件组成，大约3%的人类基因组由重复的DNA元件组成。 基因组由微卫星、1-6个DNA碱基的短串联重复序列组成。多重复序列 可以形成干扰复制和修复的替代DNA结构。这可能导致致病 重复扩增，如引起亨廷顿病，强直性肌营养不良， 以及许多脊髓小脑共济失调。结构形成重复序列内的断裂导致染色体缺失， 重排，这在经历复制应激的癌细胞中很常见。我实验室的目标是 研究由结构形成重复序列引起的基因组不稳定性的机制，并阐明细胞 进化出的防止这些有害突变的途径。我们最近发现， 结构形成CAG重复重新定位到核周边，以促进通过束的复制， 防止染色体断裂和重复扩增。这种途径依赖于蛋白质的修饰， 通过类小泛素化的复制叉，以及随后类小泛素化的蛋白质与 在S期晚期释放核孔复合物（NPC），然后在G2期释放回核内。 最近的数据表明，这一途径与几种类型的复制障碍有关，包括蛋白质 块和其他结构形成重复。因此，更好地理解这一目的至关重要。 迁移到NPC及其在促进复制和防止基因组不稳定性方面的作用，这是我们长期以来- 长期目标 我们已经开发了一个系统来跟踪扩张的CAG道或其他结构的位置- 使用显微镜在细胞核中形成重复序列，辅以生化技术检测 与重复位点相互作用的蛋白质。我们将使用芽殖酵母（S。酿酒）系统，使我们能够 联合收割机将这些方法与酵母系统的强大遗传学结合起来。酵母复制和修复 途径与人类细胞保持高水平的保守性，但酵母基因组的较小尺寸和 蛋白质组和野生型（非转化）状态的细胞是优势，这将使我们能够作出显着的 在我们的目标上取得进展。我们计划阐明重新安置停滞复制叉到核的目的 孔复合物和机制，发生在那里，以允许重新启动复制叉，使用既建立 和新颖的方法。我们还将确定核孔连接叉重启如何防止染色体 中断和重复不稳定性，并确定在fork恢复期间重复扩展如何发生。我们的目标是 了解复制体相关蛋白的NPC依赖性修饰和发生在 复制障碍，并在这样做了解重要的细胞过程，保持基因组的稳定性。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突变的策略至关重要。 发生。