Replication Of Chromosomes In Budding Yeast

摘要 染色质对于依赖于进入基因组的基本生物过程具有根本重要性。 鉴于染色质结构的多样性，一个中心问题是： 在细胞内建立和维持的这个问题可能在S阶段最重要， 染色质在新的子基因组上分解和重新组装。我们开发了新 我们已经研究了DNA复制如何在基因组中进行，以及如何在基因组中进行。 染色质建立在新生DNA上。我们最近发表的研究表明，核小体 在新复制的DNA上迅速组织起来，但这是如何发生的机制尚不清楚。 在这个建议中，我们将生成数据，为理解染色质如何 在芽殖酵母的S期建立结构。 在S期，染色质的忠实复制依赖于两个组蛋白库：来自亲本的组蛋白库， DNA和S期合成的DNA。“旧”和“新”组蛋白是如何被 复制体和沉积到染色质中的机制尚不清楚。最近的数据显示， 组蛋白可以不对称地分离到一个子细胞中！- 这就增加了老年人（或 新的）组蛋白携带重要的信息，帮助确定细胞的身份。为了理解这是如何 我们已经开发了一种新的检测方法，可以通过复制来跟踪亲本组蛋白。使用 本试验我们将确定组蛋白沉积是否受到蛋白质不对称性的影响， 复制叉，以及是否有特定的机制被细胞利用来调节新旧的位置。 组蛋白沉积。 本提案的最后一个方面是开发一种新技术来映射各个复制分叉的方式 在基因组中传播我们对DNA复制及其与染色质关系的理解 结构，核组织和基因转录的进展显着通过 开发全基因组分析。所有全基因组工具都利用大量细胞， 他们报告的是人口的平均数;因此，许多基本参数， 复制叉如何在染色质中前进尚不清楚。我们描述了一种新技术， 捕获和映射姐妹复制叉的位置和丰度， 复制起点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.