Chromatin Structure and Dynamics

描述（由申请人提供）：本项目的长期目标是了解染色质在真核细胞核中组装和利用的机制。染色质组装的研究将集中在ACF的生物化学和生物学功能。ACF是一种利用ATP的染色质组装因子，由Acf1和ISWI ATP酶两个亚基组成。与核心组蛋白伴侣结合，ACF介导组蛋白在DNA上的ATP依赖性沉积以及核小体周期性阵列的产生。该项目还将研究参与同源重组的Rad54的生物化学活性。Rad54与ACF的ISWI亚基密切相关，因此预计Rad54将在促进染色质中的同源重组中发挥关键作用。 具体目标如下。1.探讨ATP依赖的染色质体外组装机制。将采用生物化学和单分子方法来分析ACF介导的染色质组装。此外，将检查在不存在ACF的情况下介导ATP依赖性染色质组装的因素。2.分析ACF在果蝇体内的功能。一个空突变等位基因acf1产生的不精确切除的P元件。通过使用纯合突变果蝇，Acf 1对基因表达和染色体结构的影响将被研究。Acf1在细胞和多线染色体中的定位和功能也将被检查。3.检查Rad54介导染色质中链配对的能力。将研究Rad54和Rad51催化染色质中D环形成的机制。此外，天然Rad54，这似乎是在一个多亚基复合物，将从果蝇胚胎纯化。这些研究应该有助于我们对染色质组装以及DNA重组和修复的基本理解，因此应该提供适用于理解和治疗涉及这些过程缺陷的人类疾病的关键知识。

DESCRIPTION (provided by applicant): The long-term objective of this project is to understand the mechanisms by which chromatin is assembled and utilized in the eukaryotic nucleus. The studies of chromatin assembly will be focused on the biochemical and biological functions of ACF. ACF is an ATP-utilizing chromatin assembly factor that consists of two subunits, Acf1 and the ISWI ATPase. In conjunction with a core histone chaperone, ACF mediates the ATP-dependent deposition of histones onto DNA as well as the generation of periodic arrays of nucleosomes. This project will also examine the biochemical activities of Rad54, which is involved in homologous recombination. Rad54 is closely related to the ISWI subunit of ACF, and it is therefore anticipated that Rad54 will have a key role in facilitating homologous recombination in chromatin. The Specific Aims are as follows. 1. Investigate the mechanism of ATP-dependent chromatin assembly in vitro. Biochemical and single-molecule approaches will be employed to analyze ACF-mediated chromatin assembly. In addition, factors that mediate ATP-dependent chromatin assembly in the absence of ACF will be examined. 2. Analyze the function of ACF in vivo in Drosophila. A null mutant allele of acf1 was generated by imprecise excision of a P element. By using the homozygous mutant flies, the effect of Acf1 upon gene expression and chromosome structure will be investigated. The localization and function of Acf1 in cells and in polytene chromosomes will also be examined. 3. Examine the ability of Rad54 to mediate strand pairing in chromatin. The mechanism by which Rad54 and Rad51 catalyze D loop formation in chromatin will be investigated. In addition, native Rad54, which appears to be in a multisubunit complex, will be purified from Drosophila embryos. These studies should contribute to our fundamental understanding of chromatin assembly as well as DNA recombination and repair, and should thus provide critical knowledge that would be applicable to the understanding and treatment of human diseases that involve defects in these processes.