DNA topoisomerase I (Top1p) plays an important role in DNA replication, transcription and recombination. This enzyme catalyzes changes in DNA topology through the transient breakage and rejoining of a single strand in duplex DNA, in a reaction involving the covalent linkage of Top1p to the 3'phosphoryl end of the cleaved DNA. Top1p is also the target of the antitumor drug camptothecin (CPT), which poisons Top1p by reversibly stabilizing the covalent enzyme-DNA intermediate. During S-phase, the collision of advancing replication forks with these complexes produces the DNA lesions that signal cell cycle arrest and cell death. However, little is known about the nature of the lesions produced and the cellular processes involved in the recognition and resolution of Top1p-induced DNA damage. Less clear is the structural basis for drug and/or mutation-induced alterations in Top1p function and how perturbations in enzyme catalysis elicit a cytotoxic response. The goal of this proposal is to investigate the structural aspects of drug- and mutation-induced alterations in enzyme catalysis and the mechanisms of Top1p-mediated cell lethality in yeast. To accomplish this, the effects of active site mutations on the cleavage/religation equilibrium of Top1p in the presence and absence of CPT will be assessed. The function of N-terminal and linker domains in Top1p catalysis will also be analyzed with a series of human/yeast Top1 chimeras. In addition, the effects that limiting Topi protein clamp flexibility have on DNA strand cleavage and rotation will be assessed using a reversible disulfide bond to lock the closed clamp. The isolation of conditional yeast doa4-10 and cdc45-10 mutants with enhanced sensitivity to Top1p-induced damage suggest a functional link between ubiquitin-mediated proteolysis, checkpoint functions and the initiation of DNA replication in response to CPT. Assessing the role of Doa4p in cellular responses to Top1p-induced DNA damage will include an analysis of checkpoint functions in doa4-10 mutants and the isolation of extragenic mutants that suppress doa4-10 mutant cell sensitivity to Top1p poisons. The function of Cdc45p in response to CPT-induced DNA damage will be addressed by assessing the temporal regulation of origin firing and CPT effects on replication fork stalling and stability. Multicopy vector based DNA libraries will also be used to isolate dosage suppressors of cdc45-10 mutant cell sensitivity to toplT722A-induced lesions.
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