The objective of this proposal is to understand the physiological role of topoisomerase I in the eukaryotic nucleus. The avian system will be developed as a model to investigate the biochemical interaction between tokpoisomerase I and chromatin using a topoisomerase/DNA binding assay which has been shown to be a sensitive and quantitative measure of topoisomerase activity. We plan to use this assay to: 1) determine to what extent the topoisomerase will bind DNA in nucleosomes compared to protein free DNA of the same size and sequence; 2) establish if the affinity of topoisomerase for DNA is modified by structural proteins of the nucleosome; 3) determine if topoisomerase binds to inter -and/or intra-nucleosomal DNA; and 4) determine the location of endogenous topoisomerase in chromatin with respect to the central histone core of the nucleosome. We will also explore the role of toposoimerase I in transcription and DNA replication using technologies which allow us to identify and partially fractionate transcribing and replicating chromatin. The association of topoisomerase with segments of chromatin that are actively engaged in transcription and replication will then be analyzed using procedures that covalently couple the topoisomerase to its endogenous DNA substrate. We also plan to use the Sv-40 minichromosome as a model for cellular chromatin and the substrate for binding the purified topoisomerase. We will cross link the enzyme to the viral chromosome and use restriction enzymes and Southern hybridizations to map topoisomerase binding domains in a chromosomal setting. The primary significance of the proposed research is to advance our knowledge of the structural basis for alterations in chromatin organization which attend gene activation and DNA replication during processes such as differentiation, viral tumorigenesis and neoplasia.
