DNA REPLICATION COUPLED CHROMATIN ASSEMBLY

本项目的长期目标是了解 DNA复制和染色质组装之间的关系。该补助金旨在 研究DNA复制偶联染色质的生化机制 组装以及研究染色质的生物学功能 果蝇中的组装因子。该提案旨在继续 支持正在进行的果蝇染色质组装因子-1研究 （dCAF-1）和一个新发现的活动，复制偶联组装 因子（RCAF）。dCAF-1和RCAF都是 将新复制的DNA（相对于未复制的DNA）组装成 在体外SV 40 DNA复制试验中，有可能 RCAF是一种关键分子，它为染色质标记新复制的DNA 组装以及DNA的其他重要功能， 进入S期。具体目标如下。 1.果蝇染色质组装因子1（dCAF-1）基因的克隆与分析 1）。编码构成dCAF-1的四种多肽中的三种的cDNA 被隔离了这个目标的目的是克隆剩下的 dCAF-1的亚基，然后继续进行表征， dCAF-1的生化特性和生物学功能。 2.果蝇复制偶联组装体的纯化和克隆 因子（RCAF）。这些实验的目的是纯化 RCAF，编码RCAF的cDNA的分离，以及RCAF的合成和鉴定。 重组RCAF的纯化。 3. RCAF的体外和体内表征。这些目标 研究的目的是分析RCAF参与的生化机制， 在DNA复制偶联染色质组装以及生物学中， RCAF在果蝇体内的功能。 这些实验将有助于我们理解 以及染色质在以下过程中的作用， 转录、复制、修复和重组。这些研究 应直接适用于人类疾病的分析，包括 许多形式的癌症，涉及这些重要的染色质缺陷， 利用过程。

The long-term objective of this project is to understand the relation between DNA replication and chromatin assembly. This grant proposes to examine the biochemical mechanism of DNA replication-coupled chromatin assembly as well as to investigate the biological function of chromatin assembly factors in Drosophila melanogaster. This proposal seeks continued support of ongoing studies of Drosophila chromatin assembly factor-1 (dCAF-1) and a newly-discovered activity, replication-coupling assembly factor (RCAF). Both dCAF-1 and RCAF are specifically required for the assembly of newly-replicated DNA (relative to unreplicated DNA) into chromatin in an in vitro SV40 DNA replication assay. It is possible that RCAF is a key molecule that 'tags' newly-replicated DNA for chromatin assembly as well as for other important functions of DNA during progression through S phase. The Specific Aims are as follows. 1. Cloning and analysis of Drosophila chromatin assembly factor 1 (dCAF- 1). The cDNAs encoding three of the four polypeptides that comprise dCAF-1 have been isolated. The objective of this aim is to clone the remaining subunit of dCAF-1 and then to proceed onto the characterization of the biochemical properties and biological functions of dCAF-1. 2. Purification and cloning of Drosophila replication-coupling assembly factor (RCAF). The objectives of these experiments are the purification of RCAF, isolation of the cDNA(s) encoding RCAF, and the synthesis and purification of recombinant RCAF. 3. Characterization of RCAF in vitro and in vivo. The objective of these studies is to analyze the biochemical mechanism by which RCAF participates in DNA replication-coupled chromatin assembly as well as the biological function of RCAF in vivo in Drosophila. These experiments would contribute to our understanding of the formation of chromatin as well as the role of chromatin in processes such as transcription, replication, repair, and recombination. Thus, these studies should be directly applicable to the analysis of human disease, including many forms of cancer, that involve defects in these important chromatin- utilizing processes.