Chromatin-mediated maintenance of genomic integrity in germ cells

项目总结 DNA损伤的积累对所有生物体的基因组完整性都是一种威胁。DNA损伤可能由以下原因引起 外部来源(如辐射或某些化学物质)，也可作为代谢的结果自发发生 DNA复制中的压力或错误。这个研究领域与年龄相关的疾病和生殖直接相关。 失败了。为了防止这些情况，DNA修复途径完好无损是至关重要的。DNA损伤的修复是 对生殖细胞至关重要，生殖细胞是卵子和精子的前体。在人类中，认知缺陷或 生殖细胞对DNA损伤的反应表现为不孕和流产，也是导致 发育障碍(如唐氏综合症和自闭症谱系障碍)。有多条修复路径 已经确定对特定的dna损伤有反应，但我们对潜在的分子缺乏充分的了解。 根据细胞环境引导适当的修复途径选择的机制。这是高度 意义重大，因为修复途径的有效性不同。例如，在有性繁殖期间，只有一种类型的 修复途径可以适应适当的染色体分离所需的遗传物质的交换 以及后代的遗传多样性。在所有情况下，有效的DNA修复都需要特殊的染色质 环境，使人们能够接触到损伤，并招募适当的修复机器。我们的实验利用了 为了确定染色质重构体在生殖系中的作用，一种简便的遗传模型秀丽线虫 产生卵子或精子的分裂细胞的管道。我们发现生殖细胞中成功的DNA修复 需要核小体重塑和脱乙酰酶(NuRD)复合体，这是几种保守的蛋白质之一 对真核细胞染色体的动态调节很重要的复合体。我们的长期目标是学习如何 NuRD感知并对DNA损伤做出反应，防止有害突变的积累。线虫 是这些研究的理想选择，因为它的生命周期短(3-4天)，繁殖力强，而且有一个有利于 对大量卵子或精子进行遗传学和细胞学研究。本应用程序的目标是 是为了确定NuRD的催化亚单位let-418(人类的CHD4)如何确保DNA修复的保真度 并限制错误传递给后代。在目标1中，我们将使用基因工程菌株来 明确let-418是如何修复生殖细胞中的DNA损伤的，我们将使用高通量序列分析来 量化全基因组的突变率，以应对损伤。在目标2中，我们将定义LET-418在 促进DNA修复以应对自发形式的DNA损伤，我们将量化DNA损伤 由内源性(内部)细胞压力形成的。使用报告菌株和高分辨率显微镜，我们将 确定let-418突变胚系内源性应激升高的后果。预计我们的 研究结果还将揭示限制应激诱导的DNA损伤如何防止染色体的错误分离。 生育年龄较高的女性。一旦完成，我们的工作将提供对机械的关键见解 这是人类生殖障碍的原因，并将产生研究结果，为不孕不育的治疗策略提供参考。

PROJECT SUMMARY Accumulation of DNA damage is a threat to genomic integrity in all organisms. DNA damage can result from external sources (e.g. radiation or certain chemicals) and can also occur spontaneously as a result of metabolic stress or errors in DNA replication. This area of study is directly relevant to age-related disease and reproductive failure. To prevent these conditions, it is crucial that DNA repair pathways are intact. Repair of DNA lesions is critically important in germ cells, the precursors of egg and sperm. In humans, defects in the recognition or response to DNA damage in germ cells manifest as infertility and miscarriage and are also a cause of developmental disorders (e.g. Down Syndrome and Autism Spectrum Disorder). Multiple repair pathways have been identified that respond to specific DNA lesions, yet we lack a full understanding of the underlying molecular mechanisms that guide appropriate repair pathway selection depending on cellular context. This is highly significant, as repair pathways differ in their efficacy. During sexual reproduction, for example, only one type of repair pathway can accommodate exchanges in genetic material necessary for proper chromosome segregation and genetic diversity in offspring. In all scenarios, efficient DNA repair requires a specialized chromatin environment to enable access to lesions and to recruit appropriate repair machinery. Our experiments utilize the facile, genetic model Caenorhabditis elegans to determine the role of chromatin remodelers in the germ line, a pipeline of dividing cells that give rise to eggs or sperm. We discovered that successful DNA repair in germ cells requires the Nucleosome Remodeling and Deacetylase (NuRD) complex, one of several conserved protein complexes important for dynamic regulation of eukaryotic chromosomes. Our long-term goal is to learn how NuRD senses and responds to DNA damage and prevents the accumulation of harmful mutations. C. elegans is ideal for these studies due to its short (3-4 day) life-cycle, prolific reproduction, and a germ line conducive to genetically- and cytologically-tractable study of large numbers of egg or sperm. The objective of this application is to establish how the catalytic subunit of NuRD, LET-418 (CHD4 in humans), ensures the fidelity of DNA repair and limits errors from being transmitted to offspring. In Aim 1, we will use genetically-engineered strains to pinpoint how LET-418 repairs DNA lesions in germ cells, and we will use high throughput sequence analysis to quantify genome-wide mutation rates in response to damage. In Aim 2, we will define the role of LET-418 in promoting DNA repair in response to spontaneous forms of DNA damage, and we will quantify DNA lesions formed by endogenous (internal) cellular stress. Using reporter strains and high-resolution microscopy, we will determine the consequences of elevated endogenous stress in let-418 mutant germ lines. It is expected that our results will also inform how limiting stress-induced DNA damage prevents missegregation of chromosomes in females of advanced reproductive age. Once completed, our work will provide critical insight into the mechanistic causes of human reproductive disorders and will generate findings that inform therapeutic strategies for infertility.