9631121 Kadonaga The objective of these studies is to gain a better understanding of the mechanism of chromatin assembly. A crude Drosophila embryo extract was developed that allows the preparation of factors that mediate ATP-facilitated chromatin assembly. The crude extract was fractionated into a 56 kDa core-histone-binding protein and a partially-purified fraction termed the 'dCAF- 1 -containing fraction', which was thus designated because it contains dCAF-l, the Drosophila homologue of a previously-studied factor termed chromatin assembly factor-1 (CAF-1). cDNA encoding the 56 kDa core-histone-binding protein was isolated, and it was found that the 56 kDa protein is the Drosophila homologue of a previously-identified factor known as NAP-1. Purified, recombinant Drosophila NAP-1 (dNAP-1) was found to function with components in the dCAF- 1 fraction in the ATP-facilitated assembly of nucleosomal arrays. The dCAF-1-containing fraction was separated into dCAF-1, which was purified to ~50% homogeneity, and a novel chromatin assembly factor termed dCAF-6. These components will be used to: 1. Determine the minimal set of factors that is sufficient for the assembly of regularly-spaced nucleosomal arrays. The dCAF-6 chromatin assembly activity will be purified by using biochemical procedures and assays that are similar to those that have been employed thus far in the fractionation and purification of this novel activity. If possible, the cloning of dCAF-6 will be attempted. 2. Perform mechanistic studies of chromatin assembly with purified factors. These studies include: biochemical analysis of the reaction pathway; investigation of the ATP requirement for efficient assembly; examination of potential modifications of histones or assembly factors; and analysis of histone Hl incorporation into chromatin; investigation of potential protein-protein interactions among the assembly factors. %%% Genes are associated with an elaborate protein structural framework called chromatin. DNA is woun d in a precise fashion around spools of proteins into complexes called nucleosomes which with other proteins form chromatin. The components can be taken apart and put together to study how each component fits into the overall process. These studies will purify the factors that are needed to assemble the essential components of this process and identify the role different components play in the organization of chromatin. ***
