Chromatin Assembly and Dynamics

超出提供的空间。这个项目的长期目标是了解染色质在真核细胞中组装和利用的机制。这项建议主要侧重于ACF的生化和生物功能。ACF是一种利用三磷酸腺苷的染色质组装因子，由ACFL和ISWIATPase两个亚基组成。ACF与核心组蛋白伴侣蛋白结合，介导组蛋白在DNA上依赖于三磷酸腺苷的沉积以及核小体周期性排列的产生。为了补充ACF的分析，本项目还将检测RAD54的生化活性。RAD54与ACF的ISWI亚单位密切相关，参与同源重组。此外，RAD54与RAD51在染色质重塑中起协同作用。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关键过程中的缺陷的人类疾病的关键知识。具体目标如下。1.研究染色质依赖三磷酸腺苷的体外组装。将采用生化和单分子方法分析ACF介导的染色质组装的机制。此外，还将提纯和鉴定介导染色质组装的新的ATP依赖因子。2.分析ACF在果蝇体内的功能。ACFL的零突变等位基因是通过不精确地切除P元件而产生的。利用纯合突变果蝇，研究ACFL对染色体结构和基因表达的影响。ACFL在细胞和多线染色体中的定位和功能也将被检测。3.检测RAD54和RAD51在染色质中的功能。RAD54和RAD51在染色质重塑和链配对中的作用机制将被研究。此外，还将从果蝇胚胎中提纯可能存在于多亚单位复合体中的天然Rad54。表演网站========================================Section End===========================================

EXCEED THE SPACE PROVIDED. The long-term objective of this project is to understand the mechanisms by which chromatin is assembled and utilized in the eukaryotic nucleus. This proposal focuses primarily upon the biochemical and biological functions of ACF. ACF is an ATP-utilizing chromatin assembly factor that consists of two subunits, Acfl 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. To complement the analysis of ACF, this project will also examine the biochemical activities of Rad54. Rad54 is closely related to the ISWI subunit of ACF and is involved in homologous recombination. Moreover, Rad54 functions cooperatively with Rad51 in the remodeling of chromatin. 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 key processes. The Specific Aims are as follows. 1. Investigate the ATP-dependent assembly of chromatin in vitro. Biochemical and single-molecule approaches will be employed to analyze the mechanism of ACF-mediated chromatin assembly. In addition, novel ATP-dependent factors that mediate chromatin assembly will be purified and characterized. 2. Analyze the function of ACF in vivo in Drosophila. A null mutant allele of acfl was generated by imprecise excision of a P element. By using the homozygous mutant flies, the effect of Acfl upon chromosome structure and gene expression will be investigated. The localization and function of Acfl in cells and in polytene chromosomes will also be examined. 3. Examine the ability of Rad54 and Rad51 to function in chromatin. The mechanism by which Rad54 and Rad51 function in chromatin remodeling and strand pairing in chromatin will be investigated. In addition, native Rad54, which may be in a multisubunit complex, will be purified from Drosophila embryos. PERFORMANCE SITE ========================================Section End===========================================