Research Project
3301411
This proposal concerns DNA topoisomerase II from yeast. The enzyme is essential to cell viability, and its human counterpart is an increasingly important target for antitumor therapeutics. Studying the yeast enzyme offers the opportunity to investigate the enzyme's structure and catalytic mechanism in detail, due to the quantities of material available (tens of milligrams). Furthermore, mutated copies of the gene can readily be reintroduced into yeast cells and analyzed for interactions with their native cellular milieu. The enzyme is large (165 kD) and catalyzes a complex reaction- the passage of DNA strands through an enzyme-bridged gap in the DNA backbone. The protein's structure will be reduced to smaller domains and the catalytic mechanism will be defined by characterization of reaction intermediates. The initial results will provide the conceptual and experimental framework for a subsequent analysis in great detail. Structural domains of the protein will be identified by proteolysis. Domain organization and enzyme activity will be measured after limited proteolysis to test the requirement for polypeptide continuity. Reconstitution of active enzyme from appropriate cloned fragments will permit detailed testing of sequence requirements and may explain the requirement for ATP hydrolysis. Single crystals will be grown of the intact protein and/or domains thereof, to permit a high resolution structure determination. DNA binding sites will be mapped by a new methodology which combines the ability to cross-link protein to DNA with the ability to sequence peptides bound to DNA. Characterization of a reaction intermediate will provide stringent tests of current models for the enzyme's mechanism. Regions of the protein required for DNA relaxation will be defined by deletion analysis of the TOP2 gene. Contacts between topoisomerase II and other proteins will be identified by affinity chromatography and protein cross-linking. Additional in vivo functions of the protein will be tested by transplacement with mutated copies of TOP2. The in vitro and in vivo studies described will advance our knowledge of yeast topoisomerase II significantly. This information will have direct application to an understanding of the functions of the human enzyme, and of the importance of disrupting such functions with inhibitory drugs.
