Cdc45 Function Alters Cell Sensitivity to DNA Topoisomerase I Poisons

Eukaryotic DNA topoisomerase I (Top1) is a highly conserved enzyme that functions to manage the torsional strain of DNA during cellular processes such as transcription, replication, chromatid condensation and recombination. The enzyme binds duplex DNA and through a series of strand cleavage and religation reactions removes positive or negative supercoils relieving torsional strain. Top1 is the sole cellular target of the anticancer agent camptothecin, which stabilizes the covalent complex. CPT cytotoxicity is S-phase dependent. It has been suggested that the mechanism of this S-phase toxicity is due to the advancing replication forks either colliding with the stabilized drug-enzyme-DNA intermediate or colliding with positive supercoils that accumulate in front of the advancing forks leading to S-phase dependent lesions, inhibition of DNA replication and cell death. Despite extensive study, the exact events leading to cell death or repair of these lesions have yet to be defined. Using the genetically tractable budding yeast, S. cerevisiae, as a model system, a genetic screen was designed to isolate conditional temperature sensitive mutants that exhibit enhanced sensitivity to the CPT mimetic, Top1T722A. This genetic screen identified several recessive mutants (tah mutants) involved in a variety of cellular pathways including CDC45, DPB11, TAH11, which encode essential products for DNA replication and UBC9, which encodes an E3 SUMO ligase. The tah mutant cdc45-10 has a single amino acid substitution (G510R). These cells are hypersensitive to Top1T722A and transiently accumulate in early S-phase when shifted 36C due to a defect in Okazaki fragment maturation. These cells also exhibit a slow growth phenotype when a component of the DNA damage checkpoint, RAD9, is deleted and is synthetically lethal with another tah mutant dpb11-10 at 36C suggesting that cdc45-10 exhibits defects in DNA replication. To understand how Cdc45 functions to protect cells against Top1-induced DNA damage, the defects in cdc45-10 were characterized. We identified cdc45-10 as a hypomorphic allele and increased gene dosage of this mutant allele restored cell viability in the presence of Top1T722A at 36C, however, increased gene dosage failed to restore cell viability to the cdc45-10, dpb11-10 double mutant strain suggesting that the defects in cdc45-10 that specific to Top1T722A sensitivity are distinct from the defects in the synthetic interaction of cdc45-10 and dpb11-10. These two distinct functions of Cdc45 were supported by results obtained from further characterizing the defects in cdc45-10 using a dosage suppressor screen to identify extragenic suppressors that complement cdc45-10 cell sensitivity to Top1T722A and our attempts to epitope tag Cdc45 and cdc45-10. The cdc45-10 defects in origin firing and replication fork progression were characterized by isolating replication intermediates and resolving them using 2-D gel electrophoresis. Several lines of evidence including our report that cdc45-10 is hypersensitive to the antibiotic rapamycin (RAP), suggests a role for TOR signaling in S-phase. To investigate this possibility we isolated replication intermediates that were treated with MMS, RAP, HU or combinations of these drugs and origin firing and replication fork progression was visualized using 2-D gel electrophoresis.