Chromosome aberrations resulting from double-strand DNA breaks at a naturally occurring yeast fragile site composed of inverted ty elements are independent of Mre11p and Sae2p

Abstract

Genetic instability at palindromes and spaced inverted repeats (IRs) leads to chromosome rearrangements. Perfect palindromes and IRs with short spacers can extrude as cruciforms or fold into hairpins on the lagging strand during replication. Cruciform resolution produces double-strand breaks (DSBs) with hairpin-capped ends, and Mre11p and Sae2p are required to cleave the hairpin tips to facilitate homologous recombination. Fragile site 2 (FS2) is a naturally occurring IR in Saccharomyces cerevisiae composed of a pair of Ty1 elements separated by approximately 280 bp. Our results suggest that FS2 forms a hairpin, rather than a cruciform, during replication in cells with low levels of DNA polymerase. Cleavage of this hairpin results in a recombinogenic DSB. We show that DSB formation at FS2 does not require Mre11p, Sae2p, Rad1p, Slx4p, Pso2p, Exo1p, Mus81p, Yen1p, or Rad27p. Also, repair of DSBs by homologous recombination is efficient in mre11 and sae2 mutants. Homologous recombination is impaired at FS2 in rad52 mutants and most aberrations reflect either joining of two broken chromosomes in a "half crossover" or telomere capping of the break. In support of hairpin formation precipitating DSBs at FS2, two telomere-capped deletions had a breakpoint near the center of the IR. In summary, Mre11p and Sae2p are not required for DSB formation at FS2 or the subsequent repair of these DSBs.

Figure 1.—

Mechanisms of producing a recombinogenic DSB at an IR. The inverted repeat is shown as blue and red arrows, and each line represents a single DNA strand rather than duplicated chromatids. Labeled arrows show the positions of nuclease cleavage at the hairpin structure. The centromere is shown as a black oval. Only those broken DNA molecules containing a centromere are likely to produce a recoverable chromosome rearrangement. (A) Cruciform formation in a nonreplicating DNA molecule. Processing of the resulting structure by a resolvase would be expected to yield two hairpin-capped products that could be subsequently processed to yield uncapped broken DNA molecules. (B–D) DSBs produced by different positions of cleavage of the hairpin intermediate. We show hairpin formation associated with replication of the lagging strand. Cleavage at arrow 1 produces a capped hairpin in the acentric fragment or a centromere-containing fragment with a DSB proximal to FS2. Cleavage at arrow 2 results in a product in which the DSB is between the two elements of the inverted repeat. Cleavage at arrow 3 produces a capped hairpin or, if replication proceeds through the hairpin, results in a centromere-containing fragment with a DSB near the distal Ty element of FS2.

Figure 2.—

Classes of illegitimate diploids induced by low levels of DNA polymerase α. In our experiments, a GAL-POL1 MATα HIS4 THR4 haploid experimental strain was grown under conditions that result in low α-DNA polymerase. The strain was then mated to a tester strain (1225α) with the genotype MATα his4 thr4. Ty elements are shown as red (FS2) or gray (FS1) arrows, with the orientation of the arrow representing the orientation of the Ty element. On the basis of the phenotypes of the resulting diploids, they were classified as class 1 (His⁺ Thr⁺), class 2 (His⁻ Thr⁻), or class 3 (His⁺ Thr⁻). Subsequent analysis showed that there were two types of class 1 events. Class 1A events were a consequence of fusions between two MATα strains without observed genomic changes; class 1B events were a consequence of loss of chromosome III from the tester strain. Class 2 events reflected loss of chromosome III from the experimental strain. The subclasses of class 3 were 3A (translocations with a breakpoint at FS1 or FS2 and at a Ty or δ-element on a nonhomologous chromosome), 3B (telomere-capped terminal deletion on the right arm of III), 3C (DSB on the right arm of III of the experimental strain, followed by repair from the homolog in the tester strain), 3D (deletion fusing MAT and HMR), and 3E (complex rearrangement with the FS2-centered palindrome described further in the text).

Figure 3.—

Illegitimate matings in strains with mre11 mutations: plate tests of legitimate and illegitimate mating. The MATα wild-type parent strain (MS71) and isogenic GAL-POL1, GAL-POL1 mre11Δ, and GAL-POL1 mre11-H125N (a mutation eliminating the endonuclease activity of Mre11p; Moreau et al. 1999) strains were streaked on medium containing low levels of galactose (0.005%) (resulting in low levels of DNA polymerase α) and grown overnight. These strains were then mated by replica plating to four tester strains: 1225 MATa, 1225 MATα, mre11Δ MATα, and mre11-H125N MATα. After the strains were allowed to mate overnight, they were replica plated to medium on which only diploids were capable of growth.

Figure 4.—

Physical analysis of DSB formation at FS2 in strains deficient for various nucleases. All strains were grown overnight in high galactose medium and then washed in water and resuspended in medium lacking galactose for 6 hr. DNA was extracted and chromosomal DNA molecules were separated by gel electrophoresis as described in materials and methods. The separated molecules were examined by Southern analysis, using a probe derived from the left end of III. The ratio of chromosome III molecules broken at FS2 (180-kb fragment) vs. the intact III (330 kb) was quantitated using a PhosphoImager. MS71 is a wild-type haploid strain, and GAL-POL1 ty1Δ is isogenic with the GAL-POL1 strain except that it lacks one of the two Ty1 elements that compose FS2 (Lemoine et al. 2005).

Figure 5.—

Physical analysis of a translocation produced by a BIR event between nonallelic Ty elements in an mre11Δ/ mre11Δ illegitimate diploid. (A) Microarray analysis. DNA was isolated from a class 3 illegitimate diploid (DAMC560) resulting from the mating of two MATα mre11Δ strains. This sample was labeled with a Cy5 fluorescent nucleotide and mixed with a control DNA sample labeled with a Cy3 fluorescent nucleotide, and this mixture was used a hybridization probe of a microarray containing all of the yeast ORFs and intergenic regions. The ratios of hybridization are indicated as vertical lines with deletions and additions in the experimental strain shown in green and red, respectively (analysis by the CGH-Miner program). No changes were observed on chromosomes other than III and XV. The deletion breakpoint on III is at FS2, and the amplification breakpoint on XV is at YOLWTy1-1. Large gray rectangles represent Ty elements, short gray arrowheads show δ-elements, and small black arrows represent PCR primers. (B) Mechanism for generating the III–XV translocation by BIR. Centromeres are indicated by black circles, left and right telomeres are identified by labeled rectangles, and Ty elements are indicated by arrows. (C) Confirmation of translocation by PCR. The positions of the primers are shown in A. MS71 is the wild-type parental haploid from which all GAL-POL1 experimental strains are derived, and 1225 is the wild-type parental haploid from which all mating-type tester strains are derived. MS71 has the centromere-distal Ty at FS2 that 1225 lacks. As expected, PCR using primers from III (tQup) and XV (205) generates a product when DNA from the strain with the III–XV translocation is used as a template.

Figure 6.—

Analysis of a chromosome rearrangement with a 20-kb palindrome centered on FS2 in an mre11−H125N/mre11−H125N illegitimate diploid (DAMC476). (A) Microarray analysis. The analysis was performed as described in Figure 5. On chromosome III, there was a deletion distal to FS2 and a duplication of the sequences between FS1 and FS2. The region of chromosome XIV distal to YNLWTy1-2 was duplicated. Southern analysis (described in File S1) indicated the presence of a chromosome with a large palindrome centered on FS2. (B) Mechanism for generating a rearranged chromosome with an extended palindrome. Centromeres are indicated by black circles, left and right telomeres by labeled rectangles, and Ty elements by arrows.