Abstract
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.
Highlights
DNA topology via the formation of an enzyme-DNA covalent complex that is reversibly stabilized by the antitumor drug, camptothecin (CPT)
Doa4p, Sla1p, and Sla2p Affect Cell Sensitivity to DNA Topoisomerase I Poisons—To investigate cellular responses to Top1p poison-induced DNA lesions, a yeast genetic screen was performed to isolate ts mutants with enhanced sensitivity to CPT
Genetic and functional interactions between SLA1 and SLA2 have been reported [45]; our results suggest Doa4p shares an essential function with Sla1p and Sla2p
Summary
MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::HIS3 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, cdc MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sla MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sla, rho0 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sla MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sla MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, rho0 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, cdc MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sla MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sla, rho0 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sla MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa4⌬::URA3 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa4⌬::URA3, rho0 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, rad9⌬::hisG MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, rad9⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sml1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sml1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, tdp1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, tdp1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sla1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa4⌬::URA3, sla1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sla1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, rad17⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, rad17⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, chk1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, chk1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, tof1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, tof1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, tel1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, tel1⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, rad24⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, rad24⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sml1⌬::HIS3, mec1⌬::KAN MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sml1⌬::his5ϩ, mec1⌬::KAN MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, sml1⌬::HIS3, rad53⌬::KAN MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, sml1⌬::his5ϩ, rad53⌬::KAN MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, DOA4-HA MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa4-10-HA MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, vps4⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, doa, vps4⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, ubp5⌬::his5ϩ MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, ubp11⌬::TRP1 MATa, ura, his3⌬200, leu2⌬1, trp1⌬63, top1⌬::TRP1, ubp12⌬::his5ϩ. Hypomorphic alleles of CDC45 and DBP11, isolated in our screen for top1T722A- hypersensitive (tah) mutants, induce a transient accumulation of Okazaki-sized DNA fragments and enhance cell sensitivity to Top1p-induced DNA damage [39]. These results link processive DNA replication with cellular resistance to CPT. Further studies of checkpoint mutants, such as rad9⌬, mec1⌬, and tel1⌬, suggest doa4-10-mediated alterations in the Rad9p DNA damage checkpoint These results are discussed in terms of a functional link between ubiquitin-mediated proteolysis, cortical actin organization, and cellular responses to Top1p poisons
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