Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast - PubMed (original) (raw)

Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast

Anthony Admire et al. Genes Dev. 2006.

Abstract

We report here that a normal budding yeast chromosome (ChrVII) can undergo remarkable cycles of chromosome instability. The events associated with cycles of instability caused a distinctive "sectoring" of colonies on selective agar plates. We found that instability initiated at any of several sites on ChrVII, and was sharply increased by the disruption of DNA replication or by defects in checkpoint controls. We studied in detail the cycles of instability associated with one particular chromosomal site (the "403 site"). This site contained multiple tRNA genes known to stall replication forks, and when deleted, the overall frequency of sectoring was reduced. Instability of the 403 site involved multiple nonallelic recombination events that led to the formation of a monocentric translocation. This translocation remained unstable, frequently undergoing either loss or recombination events linked to the translocation junction. These results suggest a model in which instability initiates at specific chromosomal sites that stall replication forks. Forks not stabilized by checkpoint proteins break and undergo multiple rounds of nonallelic recombination to form translocations. Some translocations remain unstable because they join two "incompatible" chromosomal regions. Cycles of instability of this normal yeast chromosome may be relevant to chromosome instability of mammalian fragile sites and of chromosomes in cancer cells.

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Figures

Figure 1.

The ChrVII assay reveals elevated chromosome loss and sectored recombinants in a rad9 mutant. (A) The ChrVII system. The two homologs are shown with their genetic markers, the position of the CAN1 gene, the centromeres (filled and open circles), and the five genetic intervals of recombination (E0 through E4). Telomeres are black triangles. A possible mechanism of chromosome loss involves loss of the bottom homolog. A possible mechanism of mitotic recombination involves an event in which the chromosomes replicate, followed by either allelic recombination between homologs to form a normal mitotic recombinant, or nonallelic recombination to generate a translocation. The structures of chromosomes in three cells and their phenotypes are shown. See text. (B) rad9 mutants have a higher frequency of chromosome loss and sectored recombinants than do RAD+ cells. Frequencies and standard deviations were determined from the average of six to 10 cultures. Absolute numbers of cells per 104 cells tested are given. Below are the frequencies normalized to that for RAD+.(C) RAD+ and rad9 cells form round and sectored colonies. Cells were plated on selective media containing canavanine without adenine. Colonies were visible after 5-7 d of incubation at 30°C. The inset shows a typical round and sectored colony.

Figure 2.

Lineage analyses of unstable ChrVIIs in genetically unstable cells. (A) The logic and methodology of the lineage analysis. Two cells (hatched and open) were plated on the selection plate containing canavanine and not containing adenine. The two cells generate a round and a sectored colony, respectively. The phenotypes of individual cells in each colony were determined as shown (see text and Materials and Methods). Because the hatched founder cell was genetically stable, it generated a round colony, and consequently colonies on the analytical plate of one phenotype. Because the open founder cell was unstable, it generated a sectored colony, and consequently colonies on the analytical plate of many different phenotypes (open, gray, black). Note that some colonies on the analytical plates contain cells with heterogeneous phenotypes. If the founder cell on the analytical plate undergoes a genetic change in the first or second cell division, the colony will contain cells of two different phenotypes in two halves of the colony (or in one-fourth and three-fourths of the colony), detectable as a “fragmented” colony (see text). (B) Phenotypes of colonies on analytical plates. The phenotypes of cells from selected colonies that gave rise to a colony on an analytical plate. Phenotypes were determined by selective replica plating. For example, all 132 cells from round colony #1 gave rise to analytical colonies with an E2 phenotype, while 51 cells from colony #4 gave rise to colonies of six different phenotypes. A total of 443 cells from nine sectored rad9 colonies were analyzed, and the absolute numbers and percent of each colony phenotype are shown. Percentages were rounded to the nearest integer. See text for more details. (C) Extended lineage analysis of unstable recombinant ChrVIIs in unstable E2 cells. In the lineage analysis to the left, an E2 cell called E2-1 gave rise to cells in which 75% had lost the chromosome and 25% had retained the chromosome. Three of those E2 cells were subjected to lineage analysis, and the results are shown. Analysis of a second E2 cell is shown on the right. See text for discussion. (D) Summary of the fates of an unstable ChrVII in a genetically unstable cell. An initial cell with two ChrVII homologs undergoes chromosome changes by multiple undefined steps to form a cell with an unstable ChrVII that has a chromosomal translocation (dasemonstrated in subsequent sections). An unstable E2 ChrVII is shown. A cell with an unstable ChrVII generate cells with further ChrVII rearrangements or loss as shown. See text for discussion.

Figure 2.

Figure 3.

Unstable cells contain a chromosome of altered size. Chromosomes were prepared from rad9 sectored colonies as described in Materials and Methods, separated on pulsefield gels, and visualized by staining with EtBr or by autoradiography after Southern hybridization. (A) Chromosomes from stable round colonies (lanes 1,3,5,7,10) and from unstable sectored colonies (lanes 2,4,6,8,9,11) were stained with EtBr (lanes _1,2,7_-11) or probed with sequences that detect ERV14 (lanes 3,4) or ADE3 (lanes 5,6). Chromosomes of altered size are indicated by arrows. (B) Chromosomes from the initial rad9 strain (wt) and from eight independent sectored rad9 colonies (lanes _1_-8). Chromosomes were stained with EtBR (left panel), or hybridized with probes that detected ERV14 (middle panel) or YGL050 (right panel). No altered chromosomes were detected in lane 1.

Figure 4.

Isolation and analysis of the DNA sequence of the recombination junction, and a model indicating how the altered chromosome was formed. (A) A PCR technique was used to generate DNA fragments that contain the breakpoint junction (see Materials and Methods). PCR fragments are shown from the original rad9 strain (lane 1) and from one rad9 sectored E2 colony (lane 2; gel images). A ladder of molecular weight standards is shown. The junction fragment is ∼2 kb in size. (B) Interpretation of the structure of the recombination junction. The normal chromosome contains LTR sequence fragments (S2, D7, S3, D11, and D12). The DNA sequence of the breakpoint junction is shown below the 403-535 chromosome. See text for details. (C) Generation of a novel HpaI restriction fragment indicates the presence of the specific 403-535 unstable chromosome. A HpaI digest of DNA from the initial rad9 strain (WT) and from two sectored E2 rad9 colonies (samples 1, 2) was hybridized with a probe to the YGL050 gene (see Fig. 3). Only the preparation in sample 2 has the Hpa1 fragment predicted by the 403-535 chromosome. (D) PCR of translocation junction. Primers (TP1, TP2 in B) were used to amplify a DNA fragment spanning the junction from genomic DNA isolated from a round colony (C) and from nine independent sectored colonies. Passages 1 and 2 are genomic DNA isolated from sectored colonies expanded to ∼109 cells (in 8 mL of minimal media without adenine and tryptophan; passage 1), and then 50 μL of each culture expanded again to ∼109 cells (in 8 mL). Control primers were to YGL044 (see Materials and Methods).

Figure 5.

Fiber FISH analysis of a novel chromosome verifies its structure. The 403-535 unstable 1200-kb chromosome was excised from a pulsefield gel and subjected to fiber FISH analysis (see Materials and Methods). All ChrVII sequences were detected using a FITC modified probe (green). (A) A rhodamine-conjugated right-end probe (to the MES1 gene) detected two signals on the chromosome fiber. (B) A centromere-linked probe (see Materials and Methods) detected a single signal on each chromosome fiber of the novel chromosome. Between 20 and 100 full-length chromosome fibers were identified in both normal and unstable chromosome preparations for each analysis (see Materials and Methods). The size of chromosomes was calculated based on DNA stretching of 2.3 kb/μM.

Figure 6.

Deletions of the ChrVII 403 E2 site decrease chromosome instability. The diagram shows sequence elements in the 403 E2 site. Indicated with symbols is the location and orientation of transcription of tRNA genes, LTR σ (open chevron) and δ (gray chevron) elements, three mitochondrial sequences (black box), two origins of replication at 393,919 and 418,930, and sites of recombination breakpoints (vertical marks). In the rad9 Δ_4_/+, the region between ERV14 and YGL050 is deleted. Frequencies and standard deviations were determined from at least six cultures for each strain. The frequencies of sectored colonies between rad9 and rad9 Δ_403_/+ are significantly different as determined by a χ2 test to the P = 0.05 confidence level. The number of sectored colonies that contained the 403-535 translocation was determined by PCR (see Materials and Methods).

Figure 7.

Cycles of chromosome instability arising from fragile sites. Two chromosome homologs are shown as a jagged line and a straight line. The DNA replication and instability of only one homolog is shown. Instability begins when DNA replication stalls at the fragile sites (*) and break unless suppressed by checkpoint proteins. Broken chromosomes generate either stable or unstable cells. Stable cells form when the broken chromosome is either lost, undergoes allelic recombination with a homolog, or undergoes nonallelic recombination to form a stable translocation, or when telomere sequences are added. Unstable cells are formed if the broken chromosome undergoes nonallelic recombination joining two fragile sites. Recombination generates either an acentric (not shown), a monocentric, or a dicentric chromosome. The dicentric chromosome may be unstable because it undergoes breakage-fusion-bridge cycle events (BFB) or because of its translocation junction (**). See text for discussion.

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Cycles of chromosome instability are associated with a fragile site and are increased by defects in DNA replication and checkpoint controls in yeast - PubMed (original) (raw)