Origin firing at repetitive sequences and genome replication

项目总结/摘要 超过一半的人类基因组是由重复的DNA序列组成的，这些序列被组织为基因贫乏， 晚期复制，转录沉默的异染色质。最近的研究发现， 在癌发生和衰老过程中重复区域的转录去抑制。这种去压抑 加速了这些区域的复制，这些区域通常复制得很晚，从而耗尽了有限的 复制资源和损害基因丰富的转录活性染色质中的复制。尽管 低水平转录和晚期复制之间的关联的重要性和普遍性， 重复序列，这种联系的机制基础仍然不清楚。 芽殖酵母中的核糖体DNA（rDNA）和铜诱导的CUP 1阵列提供了强大的 阐明这些机制的实验系统：首先，在每个位点，一个单一的操作，Sir 2 rDNA的消耗和CUP 1的铜施用，激活了转录和复制， 提供了一个简单的工具来操纵这两个过程。其次，每个rDNA和CUP 1重复序列包含一个单一的， 序列定义的复制起点;这创造了复制前的统一和可预测的定位 复合物（前RC），这是启动DNA复制所需的，和核小体，它定义了 这些前RC必须发挥作用的染色质背景。我们开发了基于测序的方法， 揭示了前RC和核小体的精确位置，无论是在这些重复阵列还是在独特的起源 在基因组中。 利用这些方法，我们发现了rDNA和CUP 1染色质的特征 可能机械地连接转录和复制过程的起源：在缺乏 在转录过程中，两个起源处的前RC都被精确定位的核小体紧密包围，而 转录降低这些位点的核小体占有率并激活复制。使用此 实验设置和我们开发的染色质分析工具，我们将确定（1）是否高 邻近前RC的核小体占据抑制复制起始和（2）核小体重塑如何 我们已经证明，在rDNA阵列上转录诱导复制所需的酶， 这种核小体施加的约束并激活复制起点。

PROJECT SUMMARY/ABSTRACT Over half of the human genome is comprised of repetitive DNA sequences organized as gene-poor, late-replicating, transcriptionally silent heterochromatin. Recent studies have discerned widespread transcriptional de-repression at repetitive regions during carcinogenesis and aging. This de-repression accelerates replication of these regions, which normally replicate late, thereby depleting limiting pools of replication resources and compromising replication in gene-rich transcriptionally active chromatin. Despite the importance and prevalence of the association between low levels of transcription and late replication at repetitive sequences, the mechanistic basis for this link remains unclear. The ribosomal DNA (rDNA) and the copper-inducible CUP1 arrays in budding yeast provide powerful experimental systems in which to elucidate these mechanisms: First, at each locus, a single manipulation, Sir2 depletion at the rDNA, and copper administration at CUP1, activates both transcription and replication, providing a simple tool to manipulate both processes. Second, each rDNA and CUP1 repeat contains a single, sequence-defined origin of replication; this creates uniform and predictable positioning of pre-replicative complexes (pre-RC), which are required for initiation of DNA replication, and nucleosomes, which define the chromatin context in which these pre-RCs must act. We have developed sequencing-based methods that reveal the precise locations of pre-RCs and nucleosomes, both at these repetitive arrays and at unique origins across the genome. Using these methods, we have discovered a feature of the chromatin at both the rDNA and CUP1 origins that may mechanistically link the processes of transcription and replication: In the absence of transcription, the pre-RCs at both origins are closely flanked by precisely-positioned nucleosomes, while transcription decreases nucleosome occupancy at these sites and activates replication. Using this experimental setup and the tools for chromatin profiling we have developed, we will determine (1) whether high nucleosome occupancy adjacent to pre-RC inhibits replication initiation and (2) how nucleosome remodeling enzymes, which we have shown to be required for transcription-induced replication at the rDNA array, relieve this nucleosome-imposed constraint and activate replication origins.