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.

National Institute of Health (NIH)
National Institute of General Medical Sciences (NIGMS)
Research Project (R01)
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Biochemistry Study Section (BIO)
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Wolfe, Paul B
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University of Washington
Schools of Medicine
United States
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Yang, Zheng; Carey, James F; Champoux, James J (2009) Mutational analysis of the preferential binding of human topoisomerase I to supercoiled DNA. FEBS J 276:5906-19
Leppard, John B; Champoux, James J (2005) Human DNA topoisomerase I: relaxation, roles, and damage control. Chromosoma 114:75-85
Carey, James F; Schultz, Sharon J; Sisson, Lisa et al. (2003) DNA relaxation by human topoisomerase I occurs in the closed clamp conformation of the protein. Proc Natl Acad Sci U S A 100:5640-5
Roy, Rupa; Trowbridge, Pamela; Yang, Zheng et al. (2003) The cap region of topoisomerase I binds to sites near both ends of simian virus 40 T antigen. J Virol 77:9809-16
Yang, Zheng; Champoux, James J (2002) Reconstitution of enzymatic activity by the association of the cap and catalytic domains of human topoisomerase I. J Biol Chem 277:30815-23
Champoux, James J (2002) A first view of the structure of a type IA topoisomerase with bound DNA. Trends Pharmacol Sci 23:199-201
Champoux, J J (2001) DNA topoisomerases: structure, function, and mechanism. Annu Rev Biochem 70:369-413