042

Isolation of Saccharomyces genes affecting sensitivity to the chemotherapeutic agent camptothecin.

DNA topoisomerase I (Top1p) relaxes supercoiled DNA by a conserved mechanism of cleaving a single DNA strand allowing rotation of the cleaved strand before religation. Single strand breaks occur via a transesterification in which the active site tyrosine of Top1p becomes covalently attached to the 3' side of the break. The chemotherapeutic agent camptothecin reversibly stabilizes this transient intermediate. Progression through S phase produces persistent double strand breaks presumably due to collision of replication forks with these intermediates. The top1T ₇₂₂ A mutant stabilizes the covalent intermediate in the absence of drug. Overexpression of this allele is cytotoxic, while expression from the endogenous TOP1 promoter produces a sublethal level of DNA damage. To identify the gene products necessary to generate or respond to this unique type of DNA damage, conditional mutations causing hypersensitivity to top1T ₇₂₂ A expression were isolated. Analysis of 6000 mutagenized cells yielded 14 recessive mutants representing 13 genes. All mutant strains tested were hypersensitive to camptothecin, establishing the T ₇₂₂ A mutant as an effective camptothecin mimetic. 11 of 13 mutants were hypersensitive to hydroxyurea suggesting specific defects in replication. Cloning and characterization of 9 genes reveal that diverse cellular components affect topoisomerase poison sensitivity including cortical actin cytoskeletal proteins, Sla1p and Sla2p; ubiquitination proteins, Doa4p and Ubc9p; and DNA replication proteins, Dpb11p and Cdc45p. These results suggest an active role of the replication fork in preventing DNA damage caused by topoisomerase poisons.

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