DNA topoisomerase I (Top1p) catalyzes changes in DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT). During S-phase, collisions with replication forks convert these complexes into cytotoxic DNA lesions that trigger cell cycle arrest and cell death. To investigate cellular responses to CPT-induced DNA damage, a yeast genetic screen identified conditional tah mutants with enhanced sensitivity to self-poisoning DNA topoisomerase I mutant (Top1T722Ap), which mimics the action of CPT. Mutant alleles of three genes, DOA4, SLA1 and SLA2, were recovered. A nonsense mutation in DOA4 eliminated the catalytic residues of the Doa4p deubiquitinating enzyme, yet retained the rhodanase domain. At 36 degrees C, this doa4-10 mutant exhibited increased sensitivity to CPT, osmotic stress, and hydroxyurea, and a reversible petite phenotype. However, the accumulation of pre-vacuolar class E vesicles that was observed in doa4Delta cells was not detected in the doa4-10 mutant. Mutations in SLA1 or SLA2, which alter actin cytoskeleton architecture, induced a conditional synthetic lethal phenotype in combination with doa4-10 in the absence of DNA damage. Here actin cytoskeleton defects coincided with the enhanced fragility of large-budded cells. In contrast, the enhanced sensitivity of doa4-10 mutant cells to Top1T722Ap was unrelated to alterations in endocytosis and was selectively suppressed by increased dosage of the ribonucleotide reductase inhibitor Sml1p. Additional studies suggest a role for Doa4p in the Rad9p checkpoint response to Top1p poisons. These findings indicate a functional link between ubiquitin-mediated proteolysis and cellular resistance to CPT-induced DNA damage.

• PMID: 14990574
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Journal Article | Research Support, Non-U.S. Gov't | Research Support, U.S. Gov't, P.H.S.

Authors

Fiorani P, Reid RJ, Schepis A, Jacquiau HR, Guo H, Thimmaiah P, Benedetti P, Bjornsti MA

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