Molecular mechanisms of replication-coupled chromatin assembly

复制偶联染色质组装的分子机制

• 批准号: 10456867
• 负责人: Dirk Remus
• 金额: $ 51.01万
• 依托单位: SLOAN-KETTERING INST CAN RESEARCH
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-09-01 至 2023-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10456867
• 关键词: Address; Affect; Biochemical; Biological Assay; Cell Proliferation; Cell division; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; Chromosomal Rearrangement; Chromosomes; Complement; Coupled; DNA; DNA Repair; DNA biosynthesis; DNA replication fork; Daughter; Defect; Epigenetic Process; Gene Expression; Generations; Genetic Transcription; Genome; Goals; Histone H3; Histones; Impairment; In Vitro; Individual; Lead; Maintenance; Malignant Neoplasms; Maps; Modification; Molecular; Molecular Chaperones; Mutation; Nucleosomes; Orthologous Gene; Pathway interactions; Pattern; Phenotype; Plasmids; Positioning Attribute; Process; Proteins; Reaction; Recycling; Reporting; Role; Saccharomyces cerevisiae; Saccharomycetales; Simian virus 40; System; Testing; Time; Transact; Yeasts; arm; chromosome replication; density; dimer; experimental study; genome integrity; in vivo; insight; next generation sequencing; novel; reconstitution; restoration; segregation; tool; transmission process; yeast protein

Summary Faithful duplication of eukaryotic chromosomes during cell division requires the accurate replication of both chromosomal DNA and its associated chromatin states. However, DNA replication results in the disassembly of nucleosomes ahead of replication forks and thus poses a significant challenge to the integrity of chromatin. Chromatin restoration following DNA replication is initiated by pathways that recycle parental histones or that assemble nucleosomes de novo from newly synthesized histones. As parental histones carry epigenetic modifications, the recycling of parental histones into the daughter genomes is of particular significance for the transmission of epigenetic information across generations. The redundancy of cellular chromatin assembly pathways and the lack of functional biochemical assay systems has limited the mechanistic study of replication-coupled chromatin assembly. We have recently reconstituted the eukaryotic DNA replication reaction using purified budding yeast proteins and yeast origin-containing plasmid templates. The system recapitulates key features of cellular DNA replication, including regulated origin firing and canonical leading and lagging strand synthesis. Parental nucleosomes are also efficiently recycled on the newly replicated DNA by core replisomes in this system. However, extended nucleosome arrays are not reestablished, presumably due to the absence of the de novo nucleosome assembly pathway. In unpublished results, we have purified the budding yeast orthologs of histone chaperones implicated in replication-coupled chromatin assembly in vivo and demonstrate their ability to coordinately assemble nucleosomes from bound histone H3-H4 and H2A-H2B dimers. Combined, the reconstituted DNA replication and nucleosome assembly reactions put us in position to reconstitute replication-coupled chromatin assembly. Aim 1 focuses on mechanisms of parental histone recycling at the replication fork. We will use our recently reconstituted chromatin replication system to test the template commitment during parental histone transfer, identify histone acceptors within the replisome, and test the role of replisome components in the process. The goal of Aim 2 is to interrogate the mechanism of parental nucleosome segregation during DNA replication in vitro. We will employ a strand-specific sequencing approach to determine the distribution pattern of parental nucleosomes to the leading and lagging arms of the fork and how this distribution is controlled by replisome proteins. We will also assess parental nucleosome positions before and after replication to identify the rules governing nucleosome positioning. In Aim3 we will focus on the reconstitution and characterization of the de novo replication-coupled nucleosome assembly pathway using biochemical, structural, and next generation sequencing approaches.

摘要 真核细胞分裂过程中染色体的忠实复制需要准确的复制 染色体DNA及其相关的染色质状态。然而，DNA复制会导致 在复制分叉之前拆解核小体，因此对 染色质的完整性。DNA复制后的染色质修复是由循环的途径启动的 亲本组蛋白或从新合成的组蛋白重新组装核小体。作为父母 组蛋白携带表观遗传修饰，亲代组蛋白循环到子代基因组中是 对于表观遗传信息在世代之间的传播具有特殊的意义。的冗余性 细胞染色质组装途径和缺乏功能性生化分析系统限制了 复制偶联染色质组装的机制研究。我们最近重组了真核生物 利用纯化的芽生酵母蛋白和酵母来源的质粒进行DNA复制反应 模板。该系统概括了细胞DNA复制的关键特征，包括受调控的原点激发 和典型的领先和滞后链合成。亲本的核小体也可以有效地循环利用 核心复制体在这个系统中新复制的DNA。然而，扩展的核小体阵列不是 重新建立，可能是由于缺乏从头开始的核小体组装途径。在……里面 未发表的结果，我们已经提纯了含组蛋白伴侣蛋白的发芽酵母同系物 体内复制偶联染色质组装及其协同组装能力 核小体来自结合的组蛋白H3-H4和H_2A-H_2B二聚体。结合在一起，重组的DNA 复制和核小体组装反应使我们处于重建复制偶联的位置 染色质组件。目的1重点研究复制叉处亲代组蛋白循环的机制。我们 将使用我们最近重组的染色质复制系统来测试模板承诺 亲代组蛋白转移，确定复制体中的组蛋白受体，并测试复制体的作用 流程中的组件。目标2的目标是询问亲本核小体的机制。 DNA体外复制过程中的分离。我们将使用特定于链的测序方法来 确定亲本核小体在前臂和后臂的分布模式 复制体蛋白是如何控制这种分布的。我们还将评估亲代核小体的位置 以确定控制核小体定位的规则。在Aim3中，我们将重点关注 从头复制偶联核小体组装途径的重组与鉴定 使用生化、结构和下一代测序方法。