Regulation of Metazoan DNA Replication by Chromatin

项目总结 在细胞增殖过程中准确复制基因组对于动物的正常发育和 动态平衡。复制的调节或保真度的破坏导致许多人类病理， 尤其是癌症。因此，对支配基因组复制的机制的完全理解是 对人类健康至关重要。在人类细胞中发现的大基因组的复制需要启动 每条染色体上数千个单独位置的双向DNA合成。正在执行此操作 关键任务需要DNA和大量蛋白质之间高度受调控的相互作用，这些蛋白质的活性 必须与其他细胞活动相协调，以便基因组的所有区域只复制一次 每一个细胞分裂一次。许多实验室经过20年的研究，发现了一种 一组42个进化上保守的多肽，足以在游离细胞中启动DNA复制 以及它们的活动如何与细胞周期相协调。然而，这些机制 确定这些因素如何以及在哪里与完整的动物细胞中的基因组相互作用，以及它们是如何 一旦它们与基因组结合，就被差异激活以启动DNA复制，这一点还不清楚。 这些过程受复制蛋白的丰度、化学成分和 染色质的相对紧凑，单个染色体大区域的折叠，以及整体 细胞核内基因组的三维结构。该领域的一个主要目标是确定如何 这些组织水平中的每一个都会在不同类型的细胞中影响基因组复制和稳定性 在发育和成体组织中。这个项目将特别关注染色质组织如何影响 动物发育过程中的基因组复制和稳定性。染色质的基本构件是 核小体，组蛋白的八聚体，由约147个碱基对的DNA组成。每种组蛋白 有一条N端的尾巴，从核小体核心突出，并受到各种化学物质的影响 修饰(如甲基化、乙酰化和磷酸化)，调节染色质组织和 从而影响基因组功能的方方面面，包括DNA复制。我们已经开发了一种方法 为果蝇设计任何所需的组蛋白尾部突变，为我们提供了操纵染色质的方法 目前在任何其他动物系统中都不存在的组织。这种遗传方法将被结合在一起 用细胞生物学和新一代DNA测序方法来确定染色质组织 调节不同细胞类型的DNA复制。

PROJECT SUMMARY Accurate replication of the genome during cell proliferation is necessary for normal animal development and homeostasis. Disruption of the regulation or fidelity of replication contributes to many human pathologies, particularly cancer. Thus, a complete understanding of the mechanisms governing genome replication is paramount to human health. Replication of large genomes like that found in human cells requires the initiation of bi-directional DNA synthesis at thousands of individual locations on each chromosome. Executing this critical task requires a highly-regulated interaction between DNA and a large set of proteins whose activity must be coordinated with other cellular events such that all regions of the genome are replicated once and only once each cell division. Two decades of research by many laboratories has resulted in the identification of a set of 42 evolutionarily conserved polypeptides that are sufficient for initiation of DNA replication in a cell free setting, as well as how their activity is coordinated with the cell cycle. However, the mechanisms that determine how and where these factors interact with the genome in an intact animal cell, and how they are differentially activated to initiate DNA replication once they bind to the genome, are not well understood. These processes are modulated by the abundance of replication proteins, the chemical composition and relative compaction of chromatin, the folding of large domains of individual chromosomes, and the overall three-dimensional architecture of the genome within the nucleus. A major goal in the field is to determine how each of these levels of organization impact genome replication and stability in different cell types during development and in adult tissues. This project will specifically focus on how chromatin organization influences genome replication and stability during animal development. The basic building block of chromatin is the nucleosome, an octamer of histone proteins encompassed by ~147 base pairs of DNA. Each histone protein has an N-terminal tail that protrudes from the nucleosome core and is subject to a variety of chemical modifications (e.g. methylation, acetylation, and phosphorylation) that modulate chromatin organization and thus influence all aspects of genome function, including DNA replication. We have developed a method in Drosophila for engineering any desired histone tail mutation, providing us a means of manipulating chromatin organization that is not currently available in any other animal system. This genetic method will be combined with cell biological and next generation DNA sequencing methods to determine how chromatin organization modulates DNA replication in different cell types.