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...
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Veröffentlicht in: | The Journal of biological chemistry 2004-05, Vol.279 (20), p.21271-21281 |
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Zusammenfassung: | 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 °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 doa4 Î 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. |
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ISSN: | 0021-9258 1083-351X |
DOI: | 10.1074/jbc.M312338200 |