Characterization of a novel histone H3 phosphorylation mark in DNA replication

DNA 复制中新型组蛋白 H3 磷酸化标记的表征

• 批准号: 7692307
• 负责人: PATRICK A GRANT
• 金额: $ 26.51万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2008
• 资助国家: 美国
• 起止时间: 2008-09-30 至 2010-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7692307
• 关键词: Affinity; Antibodies; Apoptosis; Biological Assay; Biological Markers; Budgets; CDC7 gene; Camptothecin; Cell Cycle; Cell Cycle Arrest; Cell Cycle Progression; Cell Extracts; Cell Line; Cell Nucleus; Cell division; Cell physiology; Cells; Charge; Chemicals; Chromatin; Chromatin Structure Alteration; Complex; DNA; DNA Damage; DNA Packaging; DNA biosynthesis; Data; Defect; Deletion Mutation; Dependence; Dot Immunoblotting; Epigenetic Process; Eukaryotic Cell; Evaluation; Funding; Genetic Transcription; Glutamic Acid; Goals; Grant; Growth; Height; Higher Order Chromatin Structure; Histone H3; Histones; Human; Human Genome; In Vitro; Incubated; Lead; Link; Malignant Neoplasms; Mammalian Cell; Maps; Mediating; Methyl Methanesulfonate; Mitosis; Modification; Monitor; Multiprotein Complexes; Mutate; Normal Cell; Nucleosome Core Particle; Nucleosomes; Peptides; Phase; Phosphorylation; Phosphorylation Site; Phosphotransferases; Play; Post-Translational Protein Processing; Process; Proliferating; Protamine Kinase; Proteins; Regulation; Relative (related person); Replication Licensing; Research; Research Project Grants; Resistance; Role; Serine/Threonine Phosphorylation; Side; Signal Transduction; Site; Stress; Therapeutic; Threonine; Western Blotting; Yeasts; abstracting; cancer cell; cell type; chromatin remodeling; drug sensitivity; genetic regulatory protein; histone modification; hydroxyurea; in vitro Assay; in vivo; mutant; novel; research study

Abstract DNA in the nucleus of eukaryotic cells is packaged into the higher-order chromatin structure by histone proteins. Thus, DNA templated cellular processes require alteration of chromatin structure to dynamically facilitate access to packaged DNA. In general, this is accomplished by ATP-dependent chromatin remodeling, histone exchange or chemical modification of histone proteins. Phosphorylation of serine/threonine residues in particular has been shown to function in a number of cellular processes, including transcription, mitosis, apoptosis, and sporulation. We have isolated a novel histone kinase complex in yeast containing conserved S-phase regulatory proteins. This complex is capable of phosphorylating free histones, but targets histone H3 in the context of the intact histone octamer. Our preliminary data indicate that phosphorylation maps to a previously undescribed site of histone modification that may play a critical role in histone-DNA interactions and is likely of great significance to the process of DNA replication. The goals of this proposal are to purify and fully characterize the components of the multi-protein histone kinase activity, to characterize the function of this novel epigenetic mark in vivo and verify its dependence on the kinase complex in question. We also propose to map this modification on 1% of the human genome to assess its stability, localization and its suitability as a new epigenetic marker of replicating and proliferating cell types. Given our hypothesis that this modification functions in licensing of replication, we will also compare its relative abundance across the cell cycle and its requirement for cell cycle progression. As uncontrolled cell division and DNA replication is associated with the proliferation of cancer cells we anticipate that this epigenetic mark will provide a biomarker of diseased states and will offer a novel target for cancer therapeutics.

摘要 真核细胞核中的DNA被组蛋白包装成高级染色质结构 proteins.因此，DNA模板化的细胞过程需要改变染色质结构以动态地 便于获取包装好的DNA一般来说，这是由ATP依赖的染色质完成的。 重构、组蛋白交换或组蛋白的化学修饰。磷酸化 丝氨酸/苏氨酸残基特别显示在许多细胞过程中起作用， 包括转录、有丝分裂、凋亡和孢子形成。我们分离出一种新的组蛋白激酶 酵母中含有保守的S期调节蛋白的复合物。这个复合体能够 磷酸化游离组蛋白，但在完整组蛋白八聚体的情况下靶向组蛋白H3。我们 初步数据表明磷酸化定位于组蛋白的一个以前未描述的位点， 修饰可能在组蛋白-DNA相互作用中起关键作用，并且可能对 DNA复制的过程。该提案的目标是纯化和充分表征 组分的多蛋白组蛋白激酶活性，以表征这种新的功能 表观遗传标记在体内，并验证其依赖于激酶复合物的问题。我们亦建议 将这种修饰定位在1%的人类基因组上，以评估其稳定性、定位和作为 一种新的复制和增殖细胞类型的表观遗传标记。鉴于我们的假设 修改功能的复制许可，我们也将比较其相对丰度在整个 细胞周期及其对细胞周期进程的要求。细胞分裂和DNA复制失控 与癌细胞的增殖有关，我们预计这种表观遗传标记将提供一种 疾病状态的生物标志物，并将为癌症治疗提供新的靶点。