The objective is to gain an understanding of the role of topoisomerases in chromatin structure and function. The avian system has been developed as a model to identify the catalytic sites of topoisomerases I and II at the level of DNA sequence. Technologies have been developed specifically for this purpose and with this goal in mind. These studies hinge on the demonstration that endogenous topoisomerases form a transient covalent complex with DNA (in chromatin) which can be trapped, and purified away from free protein and free DNA. The DNA in the purified complexes will be characterized using the current tools of molecular biology. Specifically, by hybridization with cloned genes (developmental or housekeeping genes) tests for enrichment of different DNA sequences coupled to topoisomerases will be carried out. High resolution mapping of catalytic sites of action of topo I and II will then be carried out to correllate alterations in DNA secondary structure with topoisomerase cleavage sites in chromatin. The mapping experiments require the production of monospecific antibodies against topoisomerases. These immunologic reagents will also be used to localize topoisomerase distribution at the cytological level using immunofluorescence with the light microscope in addition to immunoelectron microscopy with protein A-colloidal gold. A second objective is to investigate the collection of type II topoisomerases that have been isolated in a single step by affinity chromatography over novobiocin-Sepharose. An experimental scheme has been devised to search for a eukaryotic gyrase among the affinity purified activities. In addition, antibodies will be prepared against the affinity purified activities for use in immuno-selecting DNA fragments containing endogenous topoisomerase II. The DNA fragments will be identified and characterized by hybridization to cloned genes. The primary significance of the work is to advance our knowledge of the structural basis for alterations in chromatin structure which attend the temporal expression of genes during differentiation. The proposal involves the use of a very well characterized developmental system and draws on knowledge accumulated during the past decade on chromatin structure, DNA structure and gene switching during development in well defined, tractable call lineages. In addition, we are combining the extensive knowledge on this system with powerful technologies developed in this lab which allow us to study the DNA binding proteins themselves as well as the DNA binding sequence of these proteins in chromatin.
