The overall goal of the proposed research is to test a series of hypotheses which are concerned with the interaction of human topoisomerase I with DNA and which are based on the recently-solved crystal structure of enzyme-DNA complexes. The crystal structure of topoisomerese I suggests that there must be hinge points within the protein to allow the enzyme to open and close as a clamp as it releases and rebinds DNA. The location of these hinge points will be determined using a combination of site-directed mutagenesis and biochemical assays, and attempts will be made to determine the structure of the protein in the absence of DNA. Site-directed mutagenesis will be used to assess the importance of a salt bridge that connects the two opposing loops in the closed conformation. The extent to which the enzyme can slide along the DNA when locked in the closed configuration will be determined. Experiments are proposed to test whether dimerization of the protein upon DNA binding, or the presence of a second DNA binding site resembling homeodomains provide the basis for the preferential binding of the enzyme to supercoiled DNA. The linker region of the protein forms a very long coiled-coil extension that protrudes from the enzyme and has an unknown function. Using crosslinking techniques, the proximity of the linker to the bound DNA in solution will be evaluated. The effects of changing selected basic amino acids within the linker on the relaxation reaction will also be determined. The hypothesis that the linker mediates protein-protein interactions in the nucleus possibly involving higher order structures will be tested using a deletion variant lacking the linker. A series of derivatives of human topoisomerase I will be generated by mutagenesis techniques that alter the cap region of the protein to determine whether this region normally hinders or facilitates DNA rotation during the relaxation of supercoiled DNA.
