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 人类基因组的一半以上由重复的DNA序列组成，这些重复的DNA序列被组织为缺乏基因的， 后期复制，转录沉默的异染色质。最近的研究发现了广泛的 癌症发生和衰老过程中重复区域的转录抑制。这种去压抑 加速这些区域的复制，这些区域通常复制较晚，从而耗尽限制池 富含转录活性的染色质中的复制资源和折衷复制。尽管 低转录水平和晚期复制之间的相关性的重要性和流行率 尽管存在重复序列，但这种联系的机制基础仍不清楚。 萌芽酵母中的核糖体DNA(RDNA)和铜诱导的CUP1阵列提供了强大的 阐明这些机制的实验系统：首先，在每个位置，一个单一的操作，Sir2 RDNA的耗尽和CUP1的铜供应，激活了转录和复制， 提供了一个简单的工具来操作这两个过程。其次，每个rDNA和CUP1重复包含一个单一的， 序列定义的复制起点；这为复制前创建了统一且可预测的定位 启动DNA复制所需的复合体(前RC)和定义 染色质背景，这些前RC必须在其中起作用。我们已经开发了基于测序的方法 揭示前RCS和核小体在这些重复阵列和独特起始处的准确位置 在整个基因组中。 使用这些方法，我们发现了rDNA和CUP1染色质的一个特征 可以机械地将转录和复制过程联系起来的起源：在没有 转录，两个起始处的前RCS都紧密地位于精确定位的核小体的两侧，而 转录减少了核小体在这些部位的占有率，并激活了复制。使用这个 实验装置和我们开发的染色质分析工具，我们将确定(1)是否高 与前RC相邻的核小体占据抑制复制启动和(2)核小体如何重塑 我们已经证明在rDNA阵列上转录诱导复制所需的酶，释放了 这种核小体施加的限制和激活复制起始点。