Molecular mechanisms of replication-coupled chromatin assembly

总结 真核生物染色体在细胞分裂过程中的忠实复制需要精确的复制 染色体DNA及其相关的染色质状态。然而，DNA复制导致 在复制叉之前拆卸核小体，因此对核小体的复制提出了重大挑战。 染色质的完整性。DNA复制后的染色质恢复是由循环途径启动的， 亲本组蛋白或从新合成的组蛋白重新组装核小体。诸如父母 组蛋白携带表观遗传修饰，亲本组蛋白再循环到子代基因组中， 对于表观遗传信息的跨代传递具有特别重要的意义。的冗余 细胞染色质装配途径和功能性生化测定系统的缺乏限制了 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 使用纯化的芽殖酵母蛋白质和含有酵母来源的质粒的DNA复制反应 模板该系统概括了细胞DNA复制的关键特征，包括受调节的起始点发射 以及规范的前导链和滞后链合成。亲本核小体也可以有效地回收， 在这个系统中核心复制体新复制的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核小体组装途径。在 未发表的结果，我们已经纯化了涉及组蛋白伴侣的芽殖酵母直系同源物， 复制偶联染色质组装在体内，并证明他们的能力，协调组装 来自结合的组蛋白H3-H4和H2 A-H2 B二聚体的核小体。结合起来，重组的DNA 复制和核小体组装反应使我们能够重建复制偶联的 染色质装配目的1集中在复制叉的亲本组蛋白再循环机制。我们 将使用我们最近重建的染色质复制系统来测试模板的承诺， 亲本组蛋白转移，鉴定复制体中的组蛋白受体，并测试复制体的作用 过程中的组件。目的二是探讨亲本核小体的作用机制 在体外DNA复制过程中分离。我们将采用链特异性测序方法， 确定亲本核小体在分叉的前导臂和滞后臂上的分布模式， 这种分布是如何被复制体蛋白控制的。我们还将评估亲本核小体的位置 在复制之前和之后，以确定管理核小体定位的规则。在Aim 3中，我们将重点关注 从头复制偶联核小体组装途径的重建和表征 使用生物化学、结构和下一代测序方法。

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 In unpublished results, we have purified the budding yeast orthologs of histone chaperones implicated in replication-coupled chromatin assembly in vivo and demonstrate their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.