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: 8531688
• 负责人: Kelly Miguel Trujillo
• 金额: $ 11.66万
• 依托单位: UNIVERSITY OF NEBRASKA MEDICAL CENTER
• 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 2015-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8531688
• 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.

项目摘要- 我们的实验室之前已经证明，在转录过程中，组蛋白H_2B的单核苷酸基化 (H_2Bub1)对于核小体在延长的RNA聚合酶之后的有效重组是重要的 II(Pol II)。该标记是通过泛素化机制的关联以共转录方式建立的 (Rad6和Bre1)和Pol II。由Spt16和Pob3组成的组蛋白伴侣复合体FACT 酵母，促进H_2Bub1的形成，并在转录过程中辅助组蛋白的再沉积。H2Bub1是 动态的，标记被泛素蛋白酶Ubp8去除，Ubp8也与POL II一起旅行。 多条证据表明，H2Bub1/FACT关系可能对DNA也很重要 复制。首先，Spt16定位于复制的起始点，并与RNA启动酶(Pol？)结合。 第二，Pob3与复制蛋白A(RPA)相互作用，复制蛋白A是结合和保护单链DNA所必需的 在复制分叉上生成。此外，Spt16/POB3都被证明是更大的 复制体进程复合体。 我的初步数据表明，H2Bub1与DNA复制有关。具体地说，我发现H2Bub1扮演着一个角色 在恢复脱氧核糖核酸合成后，安虎阻断在S早期。我发现MCM 在HTB-K123R细胞中，解旋酶从模板上脱落，在G1-S相变时不能泛素化。在……里面 第一个具体目标是，我建议确定依赖于H2Bub1的精确复制步骤。另外，我 将确定Spt16(事实)在这一过程中的作用。最后，我将开始寻找新的表观遗传学标记 这影响了DNA复制，从而扩大了我在这个奖项的独立阶段的研究领域。 我还发现，在HTB-K123R突变体中，其他复制体组分没有有效地招募 复制的起源在S阶段。与之一致的是，S阶段的缓慢完成和缓慢的分叉 进步。这一观察结果的一种可能性是在核小体动力学中存在缺陷 复制分叉。可能H_2Bub1对核小体移位或重组是重要的。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。