Chromatin Assembly and Dynamics

点击翻译按钮获取中文摘要

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===========================================