Checkpoint and recombination pathways independently suppress rates of spontaneous homology-directed chromosomal translocations in budding yeast

Abstract

Homologous recombination between short repeated sequences, such as Alu sequences, can generate pathogenic chromosomal rearrangements. We used budding yeast to measure homologous recombination between short repeated his3 sequences located on non-homologous chromosomes to identify pathways that suppress spontaneous and radiation-associated translocations. Previous published data demonstrated that genes that participate in RAD9-mediated G₂ arrest, the S phase checkpoint, and recombinational repair of double-strand breaks (DSBs) suppressed ectopic recombination between small repeats. We determined whether these pathways are independent in suppressing recombination by measuring frequencies of spontaneous recombination in single and double mutants. In the wild-type diploid, the rate of spontaneous recombination was (3 ± 1.2) × 10^-8. This rate was increased 10-30-fold in the rad51, rad55, rad57, mre11, rad50, and xrs2 mutants, seven-fold in the rad9 checkpoint mutant, and 23-fold in the mec1-21 S phase checkpoint mutant. Double mutants defective in both RAD9 and in either RAD51, RAD55, or RAD57 increased spontaneous recombination rates by ∼40 fold, while double mutants defective in both the MEC1 (ATR/ATM ortholog) and RAD51 genes increased rates ∼100 fold. Compared to frequencies of radiation-associated translocations in wild type, radiation-associated frequencies increased in mre11, rad50, xrs2, rad51, rad55 and rad9 rad51 diploid mutants; an increase in radiation-associated frequencies was detected in the rad9 rad51 diploid after exposure to 100 rads X rays. These data indicate that the S phase and G₂ checkpoint pathways are independent from the recombinational repair pathway in suppressing homology-directed translocations in yeast.

Keywords: budding yeast; cell cycle checkpoints; chromosomal translocations; homologous recombination; radiation.

FIGURE 1

Sister chromatid, translocation, and heteroallelic recombination assays used in this study. Ovals represent centromeres and lines represent chromosomes. For simplicity, the left arms of the chromosomes are not included. An arrow and feathers together denote HIS3. As indicated in the bottom left of the figure, the 5′ deletion, his3-Δ5′, lacks the feather and the 3′ deletion, his3-Δ3′, lacks the arrow. The two regions of the sequence identity shared by the his3 fragments are indicated by decorated boxes; closely spaced diagonal-filled boxes indicate a region of 167 bp, and the broadly spaced diagonal line-filled boxes indicate a region of ∼300 bp. The “X” indicates where recombination occurred. (A) The his3-truncated fragments are integrated at the TRP1 locus to measure uSCR events. His⁺ recombinants resulting from unequal SCE were selected that contain HIS3 flanked by his3-Δ3′ and his3-Δ5'. (B) Homology-directed translocation events result from recombination between the same his3 fragments located each on chromosomes II and IV. Positions of the GAL1 and trp1 are shown on chromosomes II, IV, and the chromosomal translocations. (C) Homolog recombination between ade2-a and ade2-n generates ADE2. ADE2 and ade2 alleles are represented as boxes; ade2-a and ade2-n are separated by approximately 1 kb. Figure is an adaptation from Fasullo and Sun, (2008b).

FIGURE 2

Stimulation of homology-directed translocations by ionizing radiation in wild type, rad51 and rad55 diploid strains. Radiation-associated recombination frequencies (translocations) were measured after rad51 (N = 3) and rad55 (N = 2), and wild type (N = 5) diploid strains were exposed to 0, 2, 4, 6, and 8 krads X rays. Recombination frequencies (A) were plotted against radiation dose. Net recombination frequencies were calculated by subtracting the spontaneous frequency from the radiation-associated frequency for each experiment (B). The average survival percentage was plotted against radiation dose (C). Shaded triangles represent the rad51 strain (YB170), shaded triangles represent the rad55 strain (YB744), and shaded diamonds represent the wild-type strain (YB110). Fold change was calculated by dividing 8 krad-associated frequency by the spontaneous frequency in (A).

FIGURE 3

Stimulation of homology-directed translocations by UV and ionizing radiation in wild-type, mre11, rad50, and xrs2 diploid strains. In panel (A), UV-associated recombination frequencies (translocations) were measured after wild type (N = 2), mre11 (N = 4), rad50 (N = 2), and xrs2 (N = 3) diploid strains were exposed to 60, 90, 120 and 150 J/m² UV. Net recombination frequencies were calculated by subtracting the spontaneous frequency from the radiation-associated frequency for each experiment (B). The average survival percentage was plotted against radiation dose (C). X-ray associated recombination were measured after wild type (N = 2), mre11 (N = 4), rad50 (N = 2), and xrs2 (N = 3) diploid strains were exposed 0, 2, 4, and 8 krads X rays (Panel D). Recombination frequencies (D) were plotted against radiation dose. Net recombination frequencies were calculated by subtracting the spontaneous frequency from the radiation-associated frequency for each experiment (E). The average survival percentage was plotted against radiation dose (F). Filled diamond represents the mre11 strain (YB743), filled triangle represents the rad50 strain (YB746), and the filled box represents the xrs2 strain (YB747), and the star represents the wild-type strain. (YB348). Fold change was calculated by dividing radiation-associated frequency by the spontaneous frequency in (A, D).

FIGURE 4

Stimulation of homology-directed translocations by “low dose” ionizing radiation in wild-type, rad9, rad51, and rad9 rad51 diploid strains. Radiation-associated recombination frequencies (translocations) were measured after wild type, rad51 (N = 3), rad9 (N = 3) and rad9 rad51 (N = 3) diploid strains were exposed to 100, 200, 500, 1,000 rads X rays. Recombination frequencies (A) were plotted against radiation dose. Net recombination frequencies were calculated by subtracting the spontaneous frequency from the radiation-associated frequency for each experiment (B). The average survival percentage was plotted against radiation dose (C). Filled triangle is the rad51 strain (YB170), filled box is the rad9 strain (YB134), filled diamond is the wild-type strain (YB110), and star is rad9 rad51 (YB749). Fold change was calculated by dividing 1 krad-associated frequency by the spontaneous frequency in (A).

FIGURE 5

A proposed model of how defective gap repair in rad51 and rad9 mutants could enhance ectopic recombination between his3 fragments, resulting in the generation of a nonreciprocal translocation involving chromosomes II and IV. Each line represents a single strand of DNA, and the arrow indicates the 3′end. Chromosomes II and IV are designated by roman numerals. (Left) The DSB occurs at a site on the sister chromatid after polymerase progression and initiates SCE by gap repair. RAD51 and RAD9 function in redundant pathways to promote recombinational repair and suppress ectopic recombination. (Right) The DSB occurs because of a collapsed replication fork are after replication fork regression. Replication is then reinitiated after the 3′ end of the broken chromatid invades the intact sister chromatid. BIR can generate non-reciprocal translocations if the 3′ end of the broken chromatid invades an intact nonhomologous chromosome. The dashed arrow indicates the possibility that a DSB, which cannot be repaired by gap repair in a rad51 mutant, can initiate BIR and generate a nonreciprocal translocation. DSBs occurring in S phase require RAD9 function to efficiently initiate repair of collapsed forks. The figure was adapted from Fasullo et al. (2010).