Chronic oxidative DNA damage due to DNA repair defects causes chromosomal instability in Saccharomyces cerevisiae

Abstract

Oxidative DNA damage is likely to be involved in the etiology of cancer and is thought to accelerate tumorigenesis via increased mutation rates. However, the majority of malignant cells acquire a specific type of genomic instability characterized by large-scale genomic rearrangements, referred to as chromosomal instability (CIN). The molecular mechanisms underlying CIN are not entirely understood. We utilized Saccharomyces cerevisiae as a model system to delineate the relationship between genotoxic stress and CIN. It was found that elevated levels of chronic, unrepaired oxidative DNA damage caused chromosomal aberrations at remarkably high frequencies under both selective and nonselective growth conditions. In this system, exceeding the cellular capacity to appropriately manage oxidative DNA damage resulted in a "gain-of-CIN" phenotype and led to profound karyotypic instability. These results illustrate a novel mechanism for genome destabilization that is likely to be relevant to human carcinogenesis.

FIG. 1.

Elevated levels of large-scale chromosomal aberrations in DNA excision repair-defective strains harboring elevated levels of oxidative DNA damage as measured by illegitimate-mating assays. Frequencies of each type of event were measured as described in Materials and Methods. Medians of the frequencies are indicated below the graph. Confidence limits (95%) are shown as black vertical lines. WT, wild type.

FIG. 2.

DNA excision repair and ROS-scavenging pathway interactions result in severe growth defects in BER⁻/NER⁻ tsa1 haploids. Shown is a representative tetrad dissection of the hDNP24 diploid strain (see Materials and Methods). Squares and circles indicate identified BER⁻/NER⁻ and BER⁻/NER⁻ tsa1 haploids, respectively.

FIG. 3.

Sensitivities of strains with compromised DNA excision repair and ROS scavenging to DNA-damaging agents. Equal numbers of cells were serially diluted (five times) and spotted onto rich growth medium (A) or medium containing 50 mM hydroxyurea (HU) (B), 0.005% methyl methanesulfonate (C), or 5 mM hydrogen peroxide (D). DNA repair (BER⁻ and/or NER⁻) and ROS-scavenging (tsa1Δ) backgrounds are indicated adjacent to the rows of cell dilutions.

FIG. 4.

Replicative aging-induced CIN in different DNA repair backgrounds (haploid cells). (A) Outline of experimental strategy (see the text for details). WT, wild type. (B) Ethidium bromide-stained CHEF gels separating yeast chromosomes at passage 0 (upper gel) and passage 5 (lower gel). White arrows indicate heterogeneity in chromosome sizes in founder cell lineages of BER⁻/NER⁻ strains (preexisting heterogeneity); white arrowheads indicate changes acquired by BER⁻ lineages after five passages; black arrows indicate changes acquired by BER⁻/NER⁻ lineages after five passages.

FIG. 5.

Chromosome II is highly susceptible to large-scale genomic rearrangements. (A) Negative image of ethidium bromide-stained CHEF gel for representative strains with increased (green strain designations) and decreased (red strain designations) mobility of chromosome II (red arrows) compared to that of the wild type (black strain designations). Separated chromosomes are indicated by the corresponding roman numerals. Chromosomes were resolved from largest to smallest (left to right). (B) Schematic depiction of the results of CGH analysis for rearrangements of chromosome II for the corresponding (adjacent) strains (LCH 613 through LCH 34) listed in panel A. Small black vertical bars represent unchanged open reading frames (ORFs), green vertical bars indicate the deletion of ORFs, and red vertical bars indicate the amplification of ORFs. A black rectangle encloses the region of rearrangements on chromosome II. (C) Breakpoints of rearrangements within the segment of chromosome II as detected by CGH. Red horizontal pointed bars represent ORFs as annotated in the SGD (http://www.yeastgenome.org/), and white horizontal pointed bars represent repetitive sequences. The names of the ORFs are indicated in black capital letters. Black vertical arrows indicate the breakpoints of deletions (corresponding strains are listed in green) and amplifications (corresponding strains are listed in red). The genotypes of the strains are described in Table 1.

FIG. 6.

Chromosome II in parental strains snd701 and hDNP223 contains an amplified DNA segment. (A) Negative image of an ethidium bromide-stained CHEF gel with resolved chromosomes from different wild-type strains and haploid progeny of hDNP223 containing rearranged forms of chromosome II. The red horizontal arrow indicates the position of chromosome II. Roman numerals to the left of the gel image indicate the positions of corresponding chromosomes. Numbers to the right of the gel image indicate the annotated size (in kilobase pairs) of the chromosomes as reported in the SGD (http://www.yeastgenome.org/). Strain designations are color coded as described in the legend to Fig. 5A. (B) Southern blot of the gel shown in panel A hybridized with probes specific to the HIR1 (chromosome II) (Fig. 5C) and CSM1 (chromosome III) genes. The upper horizontal arrow indicates the position of chromosome II (revealed by the HIR1 probe), and the lower horizontal arrow indicates the position of chromosome III (revealed by the CSM1 probe). Ratios of hybridized probe densities (probe II/probe III ratios) are indicated at the bottom of the panel.

FIG. 7.

Model for formation of the hot spot of large-scale rearrangements on chromosome II. Two consecutive recombination events lead to the duplication of a chromosome II segment on both sides of the centromere. (I) An unequal crossing over between an unannotated delta element (deltaX; red horizontal boxed arrow) located on the left arm of chromosome II and homologous delta14, located on the right arm of chromosome II, yields an unstable dicentric chromosome. (II) Nonhomologous end joining between YBR009C and delta10 removes one of the centromeres (black circles) and stabilizes the chromosome. The deletion of the left-side centromere (gray zigzag arrows in event I diagram) produces a tandem duplication of segment A residing on one side of the centromere. Rearrangements involving such repeats would always result in the deletion or amplification of segment C, and the breakpoint of rearrangements would be located within repetitive elements. Mapping of the breakpoint of rearrangements within nonrepetitive sequences (e.g., for isolates LCH 270 and LCH 279) (Fig. 5C) provides strong support that the hot spot of rearrangements is a consequence of the deletion of the right-side centromere (black zigzag arrows in event II diagram), which results in the product shown in the blue rectangle. Repetitive elements are shown as black boxed arrows.