Structural mechanisms of chromatin assembly

项目摘要 基因组包装成染色质，对正常生长、发育和 差异化是实验室20多年来的重点。核小体是蛋白质的基本重复单位， 染色质结构，严格调节所有使用DNA作为底物的过程，包括 转录、DNA复制、DNA修复和重组。核小体组装的复杂步骤是 由组蛋白分子伴侣引导，这些分子伴侣对于防止组蛋白的异常非特异性相互作用至关重要。 proteins.负责新生组蛋白H3和H4的复制依赖性组装的关键蛋白是 H3/H4组蛋白伴侣、抗沉默功能1（Asf 1）和染色质组装因子（CAF-1）。的 增殖细胞核抗原（PCNA）靶向染色质组装到新复制的DNA的位点。我们 生物物理学和结构研究揭示了对早期生物学的意想不到的、新颖的和基本的见解。 复制依赖的染色质组装的阶段，即涉及转移的传递机制， H3/H4从Asf 1到CAF-1的二聚体，以及CAF-1与H3/H4相互作用的结构特征。而 这些组蛋白伴侣在核小体组装中的一般功能是已知的， 这些功能的分子和结构机制仍然不清楚。因此，本提案探讨了 关于H3/H4组蛋白伴侣活性的重要和未充分研究的方面的问题， 以及影响表观遗传保真度的DNA复制位点的作用机制 作为一个新发现的作用在组装单链核小体。为了回答这些问题， 生物物理和结构方法，专门用于研究组蛋白伴侣 将与细胞中的互补生物化学和分子生物学方法一起使用。的 这些研究的结果将是更好地理解这一过程中所涉及的结构机制 在核小体组装过程中，H3/H4沉积到DNA上，这些新的见解可能会告诉我们， 染色质景观建立和如何组蛋白伴侣可能被操纵的目的 在医学研究应用和人类疾病中的表观遗传调控。

Project Summary The packaging of the genome into chromatin that is essential for normal growth, development, and differentiation has been a focus of the lab for more than 20 years. Nucleosomes are the basic repeating unit of the chromatin structure that tightly regulates all of the processes that use DNA as a substrate, including transcription, DNA replication, DNA repair, and recombination. The intricate steps of nucleosome assembly are guided by histone chaperones that are critical to prevent the aberrant nonspecific interactions of histone proteins. The key proteins responsible for replication-dependent assembly of nascent histone H3 and H4 are the H3/H4 histone chaperones, Anti-silencing function 1 (Asf1) and Chromatin Assembly Factor (CAF-1). The proliferating-cell nuclear antigen (PCNA) targets chromatin assembly to sites of newly replicated DNA. Our biophysical and structural studies have revealed unexpected, novel and fundamental 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, and the architectural features of CAF-1 interactions with H3/H4. While the general functions of these histone chaperones in nucleosome assembly are known, the fundamental molecular and structural mechanisms for these functions remain obscure. As such, this proposal explores questions about important and understudied aspects of H3/H4 histone chaperone activity from the recruitment and mechanism of action at sites of DNA replication which impact the fidelity of epigenetic inheritance as well as a newly-discovered role in the assembly of single-stranded nucleosomes. To answer these questions, biophysical and structural approaches, which have been specifically developed to study histone chaperones will be used together with complementary biochemical and molecular biology approaches in cells. The outcomes of these studies will be a better understanding of the structural mechanisms involved in the process of deposition of H3/H4 onto DNA during nucleosome assembly, and these new insights may inform how chromatin landscapes are established and how the histone chaperones might be manipulated for the purpose of epigenetic regulation in medical research applications and human disease.