Origin firing at repetitive sequences and genome replication

重复序列和基因组复制的起源

• 批准号: 10356149
• 负责人: Antonio Bedalov
• 金额: $ 55.33万
• 依托单位: FRED HUTCHINSON CANCER CENTER
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-02-01 至 2024-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10356149
• 关键词: Address; Aging; Binding; CRISPR/Cas technology; Cells; Chromatin; Complex; Copper; DNA; DNA Polymerase II; DNA Replication Timing; DNA Sequence; DNA biosynthesis; DNA replication origin; Data; Data Set; Development; Enzymes; Gene Silencing; Genes; Genetic Transcription; Genome; Genome Stability; Genomic Segment; Genomics; Goals; Health; Heterochromatin; Histone Deacetylase; Histone H4; Human; Human Genome; ISWI; Individual; Licensing; Link; Location; Mediating; Metallothionein; Methods; Micrococcal Nuclease; Modeling; Molecular; Mutation; N-terminal; Nucleosomes; Peptide Initiation Factors; Positioning Attribute; Pre-Replication Complex; Prevalence; Process; RNA; Repetitive Sequence; Replication Initiation; Replication Origin; Repression; Resources; Ribosomal DNA; Role; Saccharomycetales; Site; System; Tail; Testing; Transact; Transcription Process; Transcriptional Activation; base; carcinogenesis; chromatin remodeling; density; mutant; prevent; programs; tool

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.

项目摘要/摘要 超过一半的人基因组由重复的DNA序列组成，该序列是基因贫乏的， 延迟复制，转录静音异染色质。最近的研究已经辨别了广泛的 癌变和衰老期间重复区域的转录解抑制。这种取消抑制 加速这些区域的复制，这些区域通常会迟到，从而耗尽了限制池 复制资源和富含基因的转录活性染色质中的复制。尽管有 低转录水平与晚期复制之间关联的重要性和流行率 重复序列，此链接的机理基础尚不清楚。 核糖体DNA（rDNA）和铜诱导的CUP1阵列在萌芽的酵母中提供功能强大 阐明这些机制的实验系统：首先，在每个基因座，单个操作，sir2 rDNA的耗竭，在CUP1处给药，激活转录和复制， 提供一个简单的工具来操纵这两个过程。其次，每个rDNA和CUP1重复包含一个单个， 序列定义的复制起源；这会产生统一且可预测的重复定位 启动DNA复制所必需的复合物（PRE-RC）和核小体所必需的，该复合物定义了 这些前RC必须起作用的染色质上下文。我们开发了基于测序的方法 在这些重复的阵列和独特的起源处揭示前RC和核小体的精确位置 跨基因组。 使用这些方法，我们在rDNA和CUP1上发现了染色质的特征 可以机械地将转录和复制过程联系起来的起源：在没有的情况下 转录，两个起源处的Pre-RC紧密侧翼，由精确置位的核小体，而 转录可在这些位点降低核小体占用率并激活复制。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 与RC前RC相邻的核小体占用率抑制复制起始，并且（2）核小体重塑如何 酶，我们已证明在rDNA阵列上进行转录诱导的复制所必需的酶，请缓解 这种核小体施加的约束和激活复制起源。