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.

项目摘要 - 我们的实验室以前证明，在转录过程中，组蛋白H2b的单量化 （H2BUB1）对于伸长RNA聚合酶的有效重新组装很重要 II（Pol II）。该商标是通过泛素化机制的关联共同转录建立的 （RAD6和BRE1）与Pol II。组蛋白伴侣复合物，事实，由SPT16和POB3组成 酵母，促进H2BUB1的形成，并在转录过程中有助于组蛋白重新沉积。 H2Bub1是 动态性，其标记被泛素蛋白酶UBP8去除，该蛋白蛋白酶也与Pol II一起传播。 几条证据表明，H2BUB1/事实关系对于DNA也可能很重要 复制。首先，SPT16本地化为复制的起源，并与RNA Primase（POL？）相关。 其次，POB3与复制蛋白A（RPA）相互作用，这对于结合和保护ssDNA至关重要 在复制叉上生成。此外，SPT16/POB3均显示为较大的组成部分 补充进展复合物。 我的初步数据已在DNA复制中实现了H2BUB1。具体来说，我发现H2BUB1起作用 在S期的早期HU块之后，DNA合成的恢复。我发现MCM 在G1-S相跃迁上无法泛素化的HTB-K123R细胞中的模板中解放酶从模板上掉落。在 我提出的第一个具体目的是确定取决于H2BUB1的精确复制步骤。另外，我 将定义SPT16（事实）在此过程中的作用。最后，我将开始寻找新颖的表观遗传标记 这会影响DNA的复制，以扩大我的研究领域，以供该奖项的独立阶段。 我还发现，在HTB-K123R突变体中，其他复制部分没有有效募集 在S期中复制的起源。与此相一致，是S期和慢速叉的缓慢完成 进展。该观察结果的一种可能性是，核小体动力学存在缺陷 复制叉。也许H2BUB1对于核病室的位移或重新组装很重要。因此， 第二目的主要涉及标记的动态调节及其如何影响核病动力学 在DNA复制过程中。