Structural and Functional Studies of the Histone Chaperone CAF-1

描述(由申请人提供)：将基因组打包成染色质是正常生长、发育和分化所必需的。核小体是染色质的基本重复单位，稳定和限制对DNA的访问，形成一个动态结构，严格调控所有以DNA为底物的过程，包括转录、DNA复制、DNA修复和重组。核小体组装和拆解过程在人类疾病中很重要，从多种遗传畸形和癌症中可以看出，这些畸形和癌症与形成和修改染色质结构的蛋白质的异常有关。负责组蛋白H3和H4复制依赖的核小体组装的关键蛋白是H3/H4组蛋白伴侣蛋白、抗沉默功能1(ASF1)和染色质组装因子(CAF-1)，以及针对新复制的DNA的增殖细胞核抗原(PCNA)。这些蛋白在真核生物进化过程中高度保守，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上起着中心作用。我们的生物物理和结构研究揭示了对复制依赖的染色质组装早期阶段的意想不到和有趣的见解，即涉及H3/H4的二聚体从ASF1转移到CAF-1的移交机制。ASF1-H3/H4复合体由一个ASF1分子通过H3二聚界面和H4的C末端结合到H3/H4杂二聚体上。我们最近发现，CaF-1作为一种不对称的非正则H3/H4四聚体，具有携带H3/H4的独特机制。在目标1中，将描述参与形成CAF-1和CAF-1 H3/H4复合体的相互作用。目的2研究H3/H4通过与CAF-1和携带有增殖细胞核抗原的DNA相互作用而向DNA募集的内在热力学性质。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蛋白而开发的新的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和增殖细胞核抗原向DNA募集的基本基础。

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.