Maintaining the integrity of a genome

项目摘要/摘要 为了完全掌握基因组不稳定性的分子起源，这个领域必须在分子水平上理解 着丝粒如何促进染色体的稳定传递。基因组不稳定是一种 人类的各种病理，包括癌症和生殖衰老。我们的长期目标是确定如何 进化上保守的粘附素复合体通过其在染色体中的作用来维持基因组的完整性 分离、染色体组织和双链断裂修复。姐妹染色单体凝聚力的丧失是 推测是染色体不稳定的主要原因。此应用程序的目标是生成一个 粘附素如何在单个人类着丝粒上发挥作用以实现着丝粒凝聚力的分子模型 和准确的染色体分离。中心假设是粘附素和DNA连结在一起 创造姐妹染色单体凝聚力的着丝粒独特景观，以防止染色体不稳定。这个 因此，人类着丝粒和着丝粒凝聚力的变异可能会影响各自的传递 染色体。我们将测试着丝粒特有的凝聚力必须被认为是一种基因 姐妹染色单体凝聚和分离的决定因素，以便有一个完整的模型 染色体不稳定通过两个特定的目的发生：1)发现着丝粒凝聚力的图景 个体人类着丝粒和2)检查染色体着丝粒凝聚力如何维持整倍体。 在第一个目标下，校准的配对末端芯片SEQ将被用来映射相对于动粒的粘附素结合 人类组织培养细胞中的蛋白质和人类着丝粒阵列。这一方法将得到以下补充 细胞中相同三种成分(着丝粒、动点和粘附素)的超分辨率成像，以及 将包括基于影像的着丝粒特异性凝聚力的测定。这些方法加在一起将 制作单个人类着丝粒内部和周围的线性和3D凝聚力地图。在第二个目标中 我们将研究着丝粒特有的着丝粒凝聚力模式如何阻止染色体 培养细胞和异种移植瘤组织中的错误分离事件。结果将是根本性的 着丝粒阵列为基础的凝聚力疲劳的原理和由此导致的染色体不稳定模式。这个 研究是创新的，因为它结合了关于人类着丝粒DNA阵列的最新信息，一种新的 通过凝聚力组织着丝粒DNA的工作模型，以及新的定量分子，基因组， 和成像工具，以探索着丝粒凝聚力如何实施准确的姐妹染色单体分离。这个 拟议的研究具有重要意义，因为着丝粒阵列可能是未被识别的 染色体不稳定。许多类型的癌症都与看似随机的不稳定模式有关 可能有独特的着丝粒凝聚力图谱的分子起源。此外，许多癌症是 与影响染色体分离机制的突变有关，例如粘附素。其结果是 这个项目将对染色体不稳定的潜在机制有一个更完整的了解。

Project Summary/Abstract In order to fully grasp the molecular origins of genome instability, the field must understand at a molecular level how centromeres work to promote the stable transmission of chromosomes. Genome instability underlies a variety of human pathologies, including cancer and reproductive aging. Our long-term goal is to determine how the evolutionarily conserved cohesin complex maintains genome integrity through its roles in chromosome segregation, chromosome organization, and double-strand break repair. Loss of sister chromatid cohesion is speculated to be a major contributor to chromosome instability. The objective of this application is to produce a molecular model for how cohesin operates at individual human centromeres to achieve centromeric cohesion and accurate chromosome segregation. The central hypothesis is that cohesin and DNA catenation together create centromere-unique landscapes of sister chromatid cohesion to prevent chromosome instability. The variation in human centromeres and centromeric cohesion may therefore impact the transmission of each chromosome. We will test the idea that centromere-specific cohesion must be considered as a genetic determinant of sister chromatid cohesion and segregation in order to have a complete model for how chromosomal instability occurs through two specific aims: 1) discover the landscape of centromeric cohesion at individual human centromeres and 2) examine how chromosome centromeric cohesion maintains euploidy. Under the first aim, calibrated paired-end ChIP seq will be used to map cohesin binding relative to kinetochore proteins and human centromeric arrays in human tissue culture cells. This approach will be complemented by superresolution imaging of the same three components (centromeres, kinetochores, and cohesin) in cells, and will include imaging-based determination of centromere-specific cohesion. Together these approaches will produce a linear and 3D map of cohesion within and around individual human centromeres. In the second aim we will examine how centromere-specific patterns of centromeric cohesion prevent chromosome missegregation events in cultured cells and in xenograft tumor tissue. The outcome will be fundamental principles of centromeric array-based cohesion fatigue and resulting patterns of chromosome instability. The research is innovative because it incorporates the latest information on human centromeric DNA arrays, a new working model for the organization of centromeric DNA by cohesion, and new quantitative molecular, genomic, and imaging tools to probe how centromeric cohesion enforces accurate sister chromatid segregation. The proposed research is significant because centromeric arrays may be unrecognized genetic determinants of chromosome instability. Many types of cancer are associated with seemingly random patterns of instability that may have molecular origins in unique centromeric cohesion profiles. Furthermore, many cancers are associated with mutations that impact chromosome segregation machinery, such as cohesin. The outcome of this project will be a more complete picture of the mechanisms underlying chromosomal instability.