Mechanisms of programmed chromosome breakage

计划摘要/摘要 这项研究的总体目标是了解生物体如何安全地破坏自己的DNA。DNA倍增- 链断裂(DSB)是一种高度危险的损伤，其修复不当会导致杂合性丧失 以及拷贝数的变化，导致许多精神和发育障碍，以及癌症。 尽管存在这些风险，大多数真核生物还是在它们的基因组中的一个或多个点引入了程序化的DSB 它们的生命周期。这些突变发生在体细胞和胚系中，并具有创造遗传多样性的功能， 去除不需要的DNA，并支持适应不断变化的环境。 细胞不遗余力地保持编程的DSB的安全。它们控制着DSB的位置和时间， 促进正确的修复模板选择，并利用监督机制协调DSB的形成 其他细胞过程，包括细胞周期进程。这些控制层中的任何一个都存在缺陷 基因组不稳定风险较高的生物，从而提供了对基因组不稳定的关键见解 与癌症和导致出生缺陷和不孕的染色体异常有关。 为了研究安全的DSB形成，拟议的工作集中在两个高度保守的实例上 发育诱导的DNA断裂：(1)减数分裂重组，每个减数分裂涉及数百个DSB 减数分裂生殖细胞，以及(2)核糖体DNA(RDNA)的程序性拷贝数变化，最高 真核生物中的表达基因位点。这项拟议的工作利用遗传学、分子生物学和基因组学来 调查这些过程。大部分工作是在酿酒酵母中进行的，但 RDNA拷贝数控制的保守性也在人类细胞系中进行了测试。 为了研究减数分裂双链断裂，未来5年的研究将建立在磷酸蛋白质组学的结果基础上。 筛选以解剖与DSB形成协调的许多减数分裂过程的监视网络。 实验还将确定染色体结构在使DSB热点成为热点方面的作用，以及基因组- 将开发广泛的方法来测量整个基因组的减数分裂修复模板选择。 为了分析rDNA中的拷贝数动态，研究将集中在驱动Re-Re的机制上。 临界短rDNA簇的扩展和一种新的DNA修复中间体在其中的作用 进程。此外，这项拟议的工作将调查基因变异在基因重复之间的传播。 在进化的时间尺度上和在实验室中选择的rDNA簇。 总之，这些分析将提供对发育诱导的动态的基本见解。 DSB，为了解这些内源过程如何对植物基因组可塑性做出贡献开辟了途径 健康和疾病。

Program Summary/Abstract The overall goal of this research is to understand how organisms safely break their own DNA. DNA double- strand breaks (DSBs) are highly hazardous lesions whose improper repair can cause loss of heterozygosity and copy-number variations, leading to numerous psychiatric and developmental disorders, as well as cancers. Despite these risks, most eukaryotes introduce programmed DSBs into their genomes at one or more points in their life cycle. These breaks occur in the soma and the germ line and function to create genetic diversity, remove unwanted DNA, and support adaptation to changing environments. Cells go to great lengths to keep programmed DSBs safe. They control the location and timing of DSBs, promote correct repair-template choice, and use surveillance mechanisms to coordinate DSB formation with other cellular processes, including cell-cycle progression. Defects in any of these layers of control leave organisms with a higher risk of genome instability, and thus provide key insights into the genome instability associated with cancers and the chromosomal abnormalities that lead to birth defects and infertility. To investigate safe DSB formation, the proposed work focuses on two highly conserved instances of developmentally induced DNA breakage: (1) meiotic recombination, which involves hundreds of DSBs per meiotic germ cell, and (2) programmed copy-number changes in the ribosomal DNA (rDNA), the most highly expressed gene locus in eukaryotes. The proposed work uses genetics, molecular biology, and genomics to investigate these processes. Most of the work is conducted in the yeast Saccharomyces cerevisiae, but conservation of rDNA copy-number control is also tested in human cell lines. To investigate meiotic DSBs, research over the next 5 years will build on results of a phospho-proteomics screen to dissect the surveillance network that coordinates many meiotic processes with DSB formation. Experiments will also define the role of chromosome architecture in making DSB hotspots hot, and a genome- wide approach will be developed to measure meiotic repair-template choice across the genome. To analyze copy-number dynamics in the rDNA, research will focus on the mechanisms that drive re- expansion of critically short rDNA clusters and investigate the role of a novel DNA repair intermediate in this process. In addition, the proposed work will investigate the spreading of genetic variants among repeats of an rDNA cluster over evolutionary time scales and upon selection in the laboratory. Together, these analyses will provide fundamental insights into the dynamics of developmentally induced DSBs, open avenues for understanding how these endogenous processes contribute to genomic plasticity in health and disease.