Enhanced stimulation of chromosomal translocations by radiomimetic DNA damaging agents and camptothecin in Saccharomyces cerevisiae rad9 checkpoint mutants

Abstract

Saccharomyces cerevisiae rad9 checkpoint mutants exhibit pleiotropic phenotypes, including higher frequencies of chromosome loss, radiation sensitivity, and decreased induction of DNA damage-inducible genes. We had previously shown that rad9 mutants exhibit higher frequencies of DNA damage-associated translocations but lower frequencies of DNA damage-associated sister chromatid exchange (SCE), compared to wild type. Herein, we have shown that differences between the frequencies of DNA damage-associated recombination in the rad9 mutant and wild type depend on the identity and the concentration of the DNA damaging agent. Translocation and SCE frequencies were measured in strains containing truncated his3 fragments, located either on chromosomes II and IV, or located in tandem on chromosome IV, respectively. DNA damage-associated frequencies of translocations after exposure to hydrogen peroxide (H(2)O(2)), bleomycin, phleomycin, cisplatin, and camptothecin are higher in the rad9 diploid than in wild type. However, translocation frequencies after exposure to 4-nitroquinoline 1-oxide (4-NQO) and N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) are similar in rad9 and wild-type strains. We suggest that the deficiency in triggering G(2) arrest after exposure to specific DNA damaging agents results in the higher levels of DNA damage-associated translocations in rad9 mutants.

Fig. 1.

Recombination assays used in this study. Ovals represent centromeres and lines represent chromosomes. For simplicity, the left arms of chromosomes are not included. The position and orientation of the his3 recombinational substrates are shown as present in strains used to measure (A) reciprocal translocations and (B) unequal SCE. An “X” designates potential sites of crossovers, and the resulting chromosomal rearrangement is presented. An arrow and feathers denote HIS3. As indicated on the bottom of the figure, the 5′ deletion lacks the feathers and the 3′ deletion lacks the arrow. The two regions of sequence identity shared by the his3 fragments are indicated by decorated boxes; broadly spaced diagonal lines indicate a region of ~300 bp, and tightly spaced diagonal lines indicate a region of 167 bp.

Fig. 2.

Plate assay demonstrating higher levels of DNA damage-associated translocations in a rad9 diploid compared to a wild-type diploid. (A) YB110 (wild-typediploid) + 0.2 μl of MMS. (B) YB110 (wild-typediploid) + 1 μl of H₂O₂. (C) YB134 (rad9mutant) + 0.2 μl of MMS. (D) YB134 (rad9mutant) + μl of H₂O₂.

Fig. 3.

Electrophoretic karyotype of camptothecin-associated His⁺ recombinants generated in the rad9 diploid YB134. (A) YB134 His⁻-parent. (B) His+ recombinant containing a non-reciprocal translocations. (C) His⁺ recombinant containing a reciprocal translocation. (D) His⁺ recombinant containing a reciprocal translocation. The positions of chromosomes II, IV, and translocations CEN2 :: IV and CEN4 :: V are indicated by the arrow.