DMA topoisomerase I (Topi) plays important roles in DMAreplication, transcription and recombinationand is also the target of camptothecin (CRT), FDA approved analogs of which are effective new agents in thetreatment of human cancers. CRT poisons Topi by reversibly stabilizing a covalent enzyme-DNA complex.During S-phase, the collision of replication forks with CPT-Top1-DNA adducts produces DMAlesions thatsignal cell cycle arrest and cell death. Although it is generally accepted that Topi targeted drugs induce DNAdamage in S-phase, it is clear that signaling pathways activated in response to damage ultimately dictatecellular fate. Using yeast as a model system, conserved components of the replication machinery, CDC45and DPB11(TopBP1), protect cells from Topi damage. Rapamycin-sensitive TOR signaling also protectsyeast cells from cytotoxic DNA lesions during S-phase. Our data support a model whereby TOR acts as asurvival pathway in response to genotoxic stress by maintaining replication fork stability and the dNTP poolsnecessary for error-prone translesion DNA polymerases. Thus, TOR-dependent cell survival in response toDNA damaging agents coincides with increased mutation rates, which may contribute to the acquisition ofdrug resistance.
Three specific aims are proposed to investigate conserved aspects of the replication machinery and TORsignaling that maintain cell survival in response to cytoxic agents, suca at CRT.
In Aim 1, a combination ofyeast genetics and chromatin immunoprecipitates to query high-density tiling arrays (ChlP-chip experiments)will investigate the mechanism by which rapamycin-sensitive TOR signaling maintains replication forkstability and regulates DNA damage-induced mutagenesis.
Aim 2 proposes to determine if rapamycin-sensitive mTOR signaling regulates human cell sensitivity to cyotoxic chemotherapeutics and the acquisitionof drug resistance. A DNA fiber labeling strategy will determine if rapamycin treatment affects replication forkprogression and stability in the presence of DNA damage, while the extent of DNA damage induced will bedefined by yH2AX staining. siRNA-based approaches will determine if S-phase checkpoint function isrequired for the protective function of mTOR.
In Aim 3, an analysis of synthetic lethal interactions will definepathway interactions of the conserved human DNA replication proteins, CDC45 and TopBPI, in regulatingcell sensitivity to CRT and rapamycin. These studies will provide critical insights into the function of the TORpathway in modulating cellular responses to DNA damage, while will impact the clinical development ofrapamycin in combination with topoisomerase l-targeted therapeutics. The potential to block drug-inducedmutations that confer resistance represents a unique application of rapamycins with clinical importance forthe treatment of pediatric malignancies.
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