Role of Histone H2B Ubiquitylation in DNA Replication

组蛋白 H2B 泛素化在 DNA 复制中的作用

• 批准号: 8336820
• 负责人: Kelly Miguel Trujillo
• 金额: $ 10.93万
• 依托单位: UNIVERSITY OF NEW MEXICO HEALTH SCIS CTR
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-09-21 至 2013-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8336820
• 关键词: Affect; Area; Award; Binding; Cell Cycle; Cells; Chromatin Structure; Complex; DNA Primase; DNA biosynthesis; Data; Defect; Development; Epigenetic Process; Gene Expression; Genes; Genetic; Genetic Transcription; Health; Histone H2B; Histones; Human; Lead; Link; Malignant Neoplasms; Molecular Chaperones; Nucleosomes; Peptide Hydrolases; Pharmaceutical Preparations; Phase; Phase Transition; Phosphotransferases; Play; Polymerase; Post-Translational Protein Processing; Process; RNA; RNA Polymerase II; Recruitment Activity; Regulation; Replication Origin; Role; S Phase; Staging; Travel; Tumor Suppressor Proteins; Ubiquitin; Work; Yeasts; base; cancer therapy; career; falls; helicase; histone modification; leukemia; mutant; novel; replication factor A; uncontrolled cell growth

Project Summary- Our lab had previously demonstrated that during transcription, the monoubiquitylation of histone H2B (H2Bub1) is important for the efficient reassembly of nucleosomes in the wake of elongating RNA polymerase II (Pol II). The mark is established co-transcriptionally, via the association of the ubiquitylation machinery (Rad6 and Bre1) with Pol II. The histone chaperone complex, FACT, which consists of Spt16 and Pob3 in yeast, promotes the formation of H2Bub1, and aids in histone redeposition during transcription. H2Bub1 is dynamic, with the mark being removed by the ubiquitin protease, Ubp8, which also travels with Pol II. Several lines of evidence suggest that the H2Bub1/FACT relationship might also be important for DNA replication. First, Spt16 localizes to origins of replication and associates with the RNA primase (Pol¿). Second, Pob3 interacts with Replication Protein A (RPA), which is essential for binding and protecting ssDNA generated at replication forks. In addition, both Spt16/Pob3 were shown to be components of a larger Replisome Progression Complex. My preliminary data have implicated H2Bub1 in DNA replication. Specifically, I find that H2Bub1 plays a role in the resumption of DNA synthesis following an HU block early in S-phase. I have discovered that the MCM helicase falls off the template in htb-K123R cells that cannot be ubiquitylated at the G1-S phase transition. In the first specific aim, I propose to identify the precise replication steps that are dependent on H2Bub1. Also, I will define the role of Spt16 (FACT) in the process. Lastly, I will begin my search for novel epigenetic marks that influence DNA replication so as to expand my area of study for the independent phase of this award. I have also discovered that in the htb-K123R mutant, other replisome components are not efficiently recruited to origins of replication in S-phase. Consistent with that, is a slow completion of S-phase and slow fork progression. One possibility for this observation is that there is a defect in nucleosome dynamics at a replication fork. Perhaps H2Bub1 is important for nucleosome displacement or reassembly. Therefore, the second aim deals largely with the dynamic regulation of the mark and how it influences nucleosome dynamics during DNA replication.

项目概要- 我们的实验室以前已经证明，在转录过程中，组蛋白H2 B的单泛素化， （H2 Bub 1）对于在延伸RNA聚合酶之后核小体的有效重组是重要的 II（Pol II）.该标记是通过泛素化机制的联合， (Rad6和Bre 1）与Pol II。组蛋白伴侣复合物FACT由Spt 16和Pob 3组成， 酵母，促进H2 Bub 1的形成，并有助于转录过程中组蛋白的再沉积。H2 Bub 1是 动态的，标记被泛素蛋白酶Ubp 8去除，Ubp 8也与Pol II一起旅行。 一些证据表明，H2 Bub 1/FACT关系可能对DNA也很重要。 复制的首先，Spt 16定位于复制起点，并与RNA引发酶（Pol）结合。 其次，Pob 3与复制蛋白A（RPA）相互作用，这对于结合和保护ssDNA是必需的 在复制分叉处生成。此外，Spt 16/Pob 3都被证明是一个更大的 复制体进展复合物。 我的初步数据表明H2 Bub 1参与了DNA复制。具体来说，我发现H2 Bub 1扮演了一个角色， 在S期早期HU阻断后DNA合成的恢复中。我发现MCM 解旋酶福尔斯从htb-K123 R细胞中的模板上脱落，其在G1-S期转变时不能被泛素化。在 第一个具体的目标，我建议确定精确的复制步骤是依赖于H2 Bub 1。而且我 将确定Spt 16（FACT）在该过程中的作用。最后，我将开始寻找新的表观遗传标记 影响DNA复制，从而扩大我的研究领域，为独立阶段的这个奖项。 我还发现，在htb-K123 R突变体中，其他复制体组分不能有效地募集 到S期的复制起点。与此相一致的是，S阶段的缓慢完成和缓慢分叉 进展这一观察结果的一种可能性是，核小体动力学中存在缺陷， 复制分叉。H2 Bub 1可能在核小体移位或重组中起重要作用。因此 第二个目标主要涉及标记的动态调节以及它如何影响核小体动力学 在DNA复制过程中。