Molecular mechanisms of replication-coupled chromatin assembly

总结 细胞分裂过程中真核染色体的忠实复制需要精确的复制 染色体 DNA 及其相关染色质状态。然而，DNA 复制会导致 在复制叉之前核小体的分解，从而对复制叉提出了重大挑战 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。作为父母 组蛋白携带表观遗传修饰，亲代组蛋白再循环到子代基因组中的作用是 对于表观遗传信息的代际传递具有特殊意义。的冗余度为 细胞染色质组装途径和功能性生化检测系统的缺乏限制了 复制耦合染色质组装的机制研究。我们最近重建了真核生物 使用纯化的芽殖酵母蛋白和含有酵母起源的质粒进行 DNA 复制反应 模板。该系统概括了细胞 DNA 复制的关键特征，包括受控的起源激发 以及规范的前导链和滞后链合成。亲代核小体也可以有效地回收 该系统中核心复制体新复制的 DNA。然而，扩展的核小体阵列并不 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，我们纯化了涉及组蛋白伴侣的出芽酵母直系同源物 体内复制偶联染色质组装并证明它们协调组装的能力 来自结合组蛋白 H3-H4 和 H2A-H2B 二聚体的核小体。合并后，重组DNA 复制和核小体组装反应使我们能够重建复制耦合 染色质组装。目标 1 重点关注复制叉处亲本组蛋白回收的机制。我们 将使用我们最近重建的染色质复制系统来测试模板承诺 亲代组蛋白转移，识别复制体中的组蛋白受体，并测试复制体的作用 过程中的组成部分。目标 2 的目标是探究亲代核小体的机制 DNA 体外复制过程中的分离。我们将采用链特异性测序方法 确定亲代核小体在叉的前导臂和滞后臂上的分布模式，以及 这种分布是如何由复制体蛋白控制的。我们还将评估亲代核小体位置 复制前后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建和表征 使用生化、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 In unpublished results, we have purified the budding yeast orthologs of histone chaperones implicated in replication-coupled chromatin assembly in vivo and demonstrate their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.