The deubiquitinating enzyme Doa4p protects cells from DNA topoisomerase I poisons.
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.
Department of Molecular Pharmacology, St. Jude Children's Research Hospital, 332 N. Lauderdale, Memphis, TN 38105, USA.
Paola
P
Fiorani
Robert J D
RJ
Reid
Antonino
A
Schepis
Hervé R
HR
Jacquiau
Hong
H
Guo
Padma
P
Thimmaiah
Piero
P
Benedetti
Mary-Ann
MA
Bjornsti
D002352:Carrier Proteins / Q000235:genetics; D002453:Cell Cycle / Q000502:physiology; D003598:Cytoskeletal Proteins; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005838:Genotype; D006918:Hydroxyurea / Q000494:pharmacology; D016296:Mutagenesis; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism*; D059004:Topoisomerase I Inhibitors; D043222:Ubiquitin Thiolesterase
D002352:Carrier Proteins; D003598:Cytoskeletal Proteins; C084434:DOA4 protein, S cerevisiae; D056827:Endosomal Sorting Complexes Required for Transport; D004791:Enzyme Inhibitors; C082265:SLA1 protein, S cerevisiae; D029701:Saccharomyces cerevisiae Proteins; D059004:Topoisomerase I Inhibitors; D010450:Endopeptidases; D043222:Ubiquitin Thiolesterase; D006918:Hydroxyurea