Structural and Functional Studies of the Histone Chaperone CAF-1

组蛋白伴侣 CAF-1 的结构和功能研究

• 批准号: 8919930
• 负责人: MAIR E CHURCHILL
• 金额: $ 29.62万
• 依托单位: UNIVERSITY OF COLORADO DENVER
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-09-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8919930
• 关键词: Address; Antigen Targeting; Area; Binding; Biology; Cell Nucleus; Cell Proliferation; Cells; Chromatin; Chromatin Modeling; Chromatin Structure; Collaborations; Complex; DNA; DNA Packaging; DNA Repair; DNA biosynthesis; DNA-protein crosslink; Data; Defect; Deposition; Deuterium; Dimerization; Disease; EMSA; Eukaryota; Evolution; Fluorescence Resonance Energy Transfer; Genetic; Genetic Recombination; Genetic Transcription; Genome; Goals; Growth and Development function; Hand; Histone H2A; Histone H3; Histone H4; Histones; Human; Hydrogen; Investigation; Length; Link; Malignant Neoplasms; Maps; Mass Spectrum Analysis; Mediating; Metastatic breast cancer; Methods; Molecular; Molecular Chaperones; Molecular Genetics; Nature; Nucleosomes; Pathway interactions; Physiological; Positioning Attribute; Process; Proliferating Cell Nuclear Antigen; Property; Proteins; Public Health; Recombinants; Recruitment Activity; Research; Research Personnel; Role; Saccharomyces cerevisiae; Saccharomycetales; Severities; Site; Solid Neoplasm; Staging; Structure; System; Technology; Testing; Therapeutic Intervention; Thermodynamics; Work; Yeast Model System; Yeasts; base; biophysical techniques; chromatin assembly factor I; design; dimer; fluorophore; genetic approach; human disease; in vivo; innovation; insight; interest; malformation; monomer; mutant; novel; public health relevance

DESCRIPTION (provided by applicant): The packaging of the genome into chromatin is essential for normal growth, development, and differentiation. Nucleosomes are the basic repeating unit of chromatin that stabilize and restrict access to the DNA, forming a dynamic structure that tightly regulates all of the processes that use DNA as a substrate, including transcription, DNA replication, DNA repair, and recombination. Nucleosome assembly and disassembly processes are important in human disease, as seen in the multiple genetic malformations and cancers that have been linked to aberrations in proteins that form and modify chromatin structure. The key proteins responsible for replication-dependent nucleosome assembly of histone H3 and H4 are the H3/H4 histone chaperones, Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor (CAF-1), as well as proliferating-cell nuclear antigen (PCNA), which targets these to sites of newly replicated DNA. These proteins are highly conserved throughout eukaryotic evolution and are the focus of this study because of their central role in histone H3/H4 deposition onto newly replicated DNA. Our biophysical and structural studies have revealed unexpected and interesting insights into the early stages of replication-dependent chromatin assembly, namely the hand-off mechanism involving transfer of dimers of H3/H4 from Asf1 to CAF-1. The Asf1-H3/H4 complex comprises one molecule of Asf1 bound to an H3/H4 heterodimer through the H3 dimerization interface and the C-terminus of H4. We have recently found that CAF-1 has a unique mechanism for carrying H3/H4 as an asymmetric non-canonical H3/H4 tetramer. In Aim 1, will delineate the interactions that are involved in the formation of CAF-1 and CAF-1 H3/H4 complexes. Aim 2 addresses the intrinsic thermodynamic properties of the recruitment of H3/H4 to DNA via interactions with CAF-1 and PCNA-loaded DNA. These studies take advantage of recombinant chromatin assembly factors, novel biophysical and structural approaches that have been specifically developed to study histone chaperones, as well as physiological analyses in the yeast model system. This work will elucidate a new mechanism for H3/H4 chaperone activity and explain the fundamental basis of H3/H4 recruitment to DNA via CAF-1 and PCNA.

描述（由申请人提供）：将基因组包装到染色质中对于正常生长、发育和分化至关重要。核小体是染色质的基本重复单位，可稳定并限制 DNA 的进入，形成动态结构，严格调节以 DNA 为底物的所有过程，包括转录、DNA 复制、DNA 修复和重组。核小体组装和拆卸过程在人类疾病中非常重要，正如在多种遗传畸形和癌症中所见，这些畸形和癌症与形成和修饰染色质结构的蛋白质畸变有关。负责组蛋白 H3 和 H4 复制依赖性核小体组装的关键蛋白是 H3/H4 组蛋白伴侣、抗沉默功能 1 (Asf1) 和染色质组装因子 (CAF-1)，以及增殖细胞核抗原 ( PCNA），它将它们靶向新复制的 DNA 位点。这些蛋白质在整个真核进化过程中高度保守，并且是本研究的重点，因为它们在组蛋白 H3/H4 沉积到新复制的 DNA 上的过程中发挥着核心作用。我们的生物物理和结构研究揭示了复制依赖性染色质组装早期阶段的意外且有趣的见解，即涉及 H3/H4 二聚体从 Asf1 转移到 CAF-1 的传递机制。 Asf1-H3/H4 复合物包含一个 Asf1 分子，该 Asf1 分子通过 H3 二聚化界面和 H4 的 C 末端与 H3/H4 异二聚体结合。我们最近发现 CAF-1 具有独特的机制来携带 H3/H4 作为不对称的非典型 H3/H4 四聚体。在目标 1 中，将描述参与 CAF-1 和 CAF-1 H3/H4 复合物形成的相互作用。目标 2 解决了通过与 CAF-1 和 PCNA 负载的 DNA 相互作用将 H3/H4 招募到 DNA 的内在热力学特性。这些研究利用了重组染色质组装因子、专门为研究组蛋白伴侣而开发的新型生物物理和结构方法，以及酵母模型系统中的生理分析。这项工作将阐明 H3/H4 伴侣活性的新机制，并解释 H3/H4 通过 CAF-1 和 PCNA 招募到 DNA 的基本原理。