DNA topoisomerase I (Top1) plays important roles in DNA replication, transcription and recombination, by catalyzing changes in DNA topology through a mechanism of transient DNA strand breakage and rejoining. This enzyme is also the target of camptothecin (CRT), FDA approved analogs of which are effective new agents in the treatment of human cancers. CRT poisons Top1 by reversibly stabilizing a covalent enzyme-DNA complex. During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DNA lesions that signal cell cycle arrest and cell death. Yet, little is known of the interactions with the replication machinery required for the formation of Top1-DNA damage or the nature of the lesions produced. The goals of this proposal are to investigate critical aspects of Top1 architecture and interactions with the replication machinery that dictate cellular responses to CRT in yeast and in human cells. Studies in the genetically tractable yeast support the hypothesis that interactions between conserved components of the replication machinery (CDD45, DPB11 and PCNA) and the flexible linker domain of Top1 alter the stability of the Top 1-DNA CRT covalent complex, and thereby, dictate cell sensitivity to CRT.
Three aims are proposed to test this model. (1) To investigate alterations in Top1 active site geometry and linker flexibility that affect cell sensitivity to Top1 poisons. CRT-induced chromatin association of Top1 in S-phase will be assessed in yeast chromatin immunoprecipitations (ChIP) and high density TileArrays. Using siRNA to downregulation endogenous Top1, human cell responses to CRT will be assessed by inducible expression of codon-altered Top1 mutants. These studies will define aspects of Top1 linker architecture critical for cytotoxic activity of CRT in S-phase. (2) To determine if the functional interplay between the flexible linker of Top1 and PCNA-polymerase complexes affects cell sensitivity to CRT. Mutational studies will define the Top1 interacting sites on PCNA, while the functional association of these proteins will be assessed by immunostaining, immunoprecipitation and ChIP. These studies will decipher the physical interactions with the replication machinery necessary for poisoning Top1. (3) To define the function of Cdc45 and Dpb11 in regulating DNA replication and fork progression in response to CRT in yeast and human cells. Using 2-D gels and ChlPs, the efficiency of origin firing and fork stability in response to Top1-DNA damage in yeast will be defined. A robotics-based, synthetic genetic analysis will define functional interactions with other yeast genes. siRNA studies will assess the conservation of these interactions in regulating human cell sensitivity to CRT. Given the significant interest in chemotherapeutics that target Top1 and the conservation of the replication machinery, the results of these studies will provide new insights to guide the clinical development of these agents, help define prognostic indicators of tumor response and provide new targets for drug development.
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