Replication Of Chromosomes In Budding Yeast

摘要 染色质对于依赖于获取基因组的基本生物过程至关重要。 鉴于染色质结构的多样性，一个中心问题是：染色质结构是如何的 在一个单元内建立和维护？这个问题在S阶段可能是最重要的， 染色质在新的子代基因组上分解和重新组装。我们开发了新的 我们用来研究DNA复制如何跨基因组进行以及如何进行的工具 染色质建立在新生的DNA上。我们最新发表的研究表明，核小体 都是在新复制的DNA上迅速组织起来的，但这是如何发生的机制尚不清楚。 在这项提案中，我们将生成数据来提供一个基本框架，以了解染色质是如何 在发芽酵母中，结构建立在S相。 S期染色质的忠实复制依赖于两个组蛋白池：来自亲本的组蛋白 脱氧核糖核酸和S期合成的DNA。如何处理“旧”和“新”组蛋白 复制体和沉积到染色质中还不是很清楚。最近的数据表明，旧的 组蛋白可能不对称地分离到一个子细胞！-增加了年老(或 新的)组蛋白携带有助于确定细胞身份的重要信息。为了理解这是如何 为了避免这种情况的发生，我们开发了一种新的方法，可以通过复制来跟踪父母的组蛋白。vbl.使用 在这项测试中，我们将定义组蛋白沉积是否受到组蛋白不对称性质的影响 复制分叉以及细胞是否利用特定机制来调节新旧位置 组蛋白被沉积。 这项提议的最后一个方面是开发一种新技术，以绘制单个复制分叉的情况 跨基因组进行研究。我们对DNA复制及其与染色质关系的理解 结构、核组织和基因转录通过 全基因组分析的发展。所有基因组范围的工具都利用大量的细胞群，这意味着 他们报告的是人口的平均水平；因此，许多基础参数 复制叉是如何通过染色质的尚不清楚。我们描述了一种新技术 捕获并映射从同一站点启动的姊妹复制分支的位置和数量 复制源。我们展示了如何使用这些信息来生成一个全新的视图 复制在整个基因组中进行。

Abstract Chromatin is of fundamental importance to basic biological processes that rely on access to the genome. Given that there is such diversity in chromatin structure, a central question is: how is chromatin structure established and maintained within a cell? This question is perhaps most significant during S-phase when chromatin is disassembled and reassembled on the new daughter genomes. We have developed new tools with which we have investigated how DNA replication proceeds across a genome and how chromatin is established on nascent DNA. Our most recent published work has shown that nucleosomes are rapidly organized on newly replicated DNA, but the mechanics of how this happens remain unclear. In this proposal we will generate data to provide a basic framework for understanding how chromatin structures are established in S-phase in budding yeast. Faithful duplication of chromatin during S-phase relies on two pools of histones: those from the parental DNA and those synthesized during S-phase. How the “old” and “new” histones are handled by the replisome and deposited into chromatin is not well understood. Recent data has suggested that old histones may be asymmetrically segregated to one daughter cell! – raising the possibility that old (or new) histones carry important information that help define cell identity. In order to understand how this may occur, we have developed a new assay that can track parental histones through replication. Using this assay we will define whether histone deposition is influenced by the asymmetric nature of the replication fork and if there are specific mechanisms utilized by the cell to regulate where old and new histones are deposited. The final aspect of this proposal is to develop a new technology to map how individual replication forks proceed across genomes. Our understanding of DNA replication and its relationship with chromatin structure, nuclear organization and gene transcription has advanced significantly through the development of genome-wide assays. All genome-wide tools utilize large populations of cells meaning that they report on the average of the population; as such, many of the basic parameters that underlie how a replication fork progresses through chromatin are not known. We describe a new technique that captures and maps the positions and abundance of sister replication forks that initiated from the same replication origin. We show how this information can be used to generate an entirely new view of how replication proceeds across a genome.