Genome-wide high-resolution mapping of chromosome fragile sites in Saccharomyces cerevisiae

Abstract

In mammalian cells, perturbations in DNA replication result in chromosome breaks in regions termed "fragile sites." Using DNA microarrays, we mapped recombination events and chromosome rearrangements induced by reduced levels of the replicative DNA polymerase-α in the yeast Saccharomyces cerevisiae. We found that the recombination events were nonrandomly associated with a number of structural/sequence motifs that correlate with paused DNA replication forks, including replication-termination sites (TER sites) and binding sites for the helicase Rrm3p. The pattern of gene-conversion events associated with cross-overs suggests that most of the DNA lesions that initiate recombination between homologs are double-stranded DNA breaks induced during S or G2 of the cell cycle, in contrast to spontaneous recombination events that are initiated by double-stranded DNA breaks formed prior to replication. Low levels of DNA polymerase-α also induced very high rates of aneuploidy, as well as chromosome deletions and duplications. Most of the deletions and duplications had Ty retrotransposons at their breakpoints.

Keywords: break-induced replication; loss of heterozygosity; mitotic cross-overs.

Fig. 1.

Patterns of LOH observed in one unsectored colony of WS84 (WS84-2#6). Genomic DNA was isolated from one colony formed on medium with low levels of galactose. This sample was hybridized to SNP-specific microarrays. The blue and red lines show hybridization to YJM789-specific and W303a-specific SNPs, respectively (normalized to a control strain). Note that the SGD coordinates on the x axis are in base pairs, with the left telomere representing base 1.

Fig. 2.

Low- and high-resolution analysis of LOH events on chromosome XV (WS84-2#6). (A) Low-resolution. In this depiction, we use a moving window that includes nine SNPs. Four LOH regions are evident, one terminal and three interstitial. The y axis indicates the hybridization ratio (HR). (B) High-resolution analysis of an LOH region (marked with an arrow in A). Each square or diamond represents the hybridization value to an individual oligonucleotide on the microarray.

Fig. 3.

Depictions of homologous recombination events leading to LOH in unsectored colonies of WS84. We show the paired homologs in G2, with the centromeres shown as ovals or circles. The cells with the genomic alteration detected by microarrays are outlined in rectangles. (A) Repair of a DSB on one of the red chromatids resulting in a gene conversion without an associated cross-over. The nonreciprocal transfer of sequences from the undamaged chromatid to the damaged chromatid results in an interstitial LOH region. (B) Repair of a DSB associated with a cross-over. If one of the recombinant chromatids is segregated into a cell with a nonrecombinant chromatid, a terminal region of LOH is observed. (C) Repair of a DSB by a conversion-associated cross-over. (D) Repair of a DSB by BIR. Following a DSB in one of the red chromatids, the chromosome fragment centromere-distal to the break is lost. The remaining fragment copies one of the blue chromatids. (E) Unequal sister-chromatid recombination between directly oriented repeats (arrows). The resulting cross-over generated a cell with an interstitial duplication (outlined in black) and a deletion in the other daughter cell. (F) Terminal deletions and duplications resulting from a BIR event between repeats located on nonhomologous chromosomes.

Fig. 4.

Distribution of unselected cross-over/BIR events and interstitial LOH events in strain WS84. We mapped 201 interstitial LOH events and events representing cross-overs/BIR in WS84 cells with low levels of DNA polymerase-α. Chromosomes are drawn to scale with chromosome I (the smallest) being 230 kb in size. Yellow hash marks show the location of the SNPs, and centromeres are shown as circles. Green lines indicate cross-overs and black lines show interstitial LOH events with the length of the line proportional to size of the LOH region.

Fig. 5.

Types of cross-overs and associated conversions that give rise to red/white sectored colonies in WS84 and MG47. In the diploids WS84 and MG47, the markers SUP4-o and HYG are located near the telomeres of the left arm of chromosome V and the right arm of chromosome IV, respectively. As discussed in the text, a cross-over can produce a red/white sectored colony in which the hygromycin-resistance phenotype also sectors (A–D), whereas a BIR event can produce a pink/white sectored colony (E) or a pink/red sectored colony (not shown). The position of the recombinogenic DSB is shown as a black triangle. (A) Cross-over unassociated with a gene conversion. In such sectored colonies, the transitions between heterozygous and homozygous markers are at the same position in both sides of the sector. (B) Cross-over associated with repair of a single broken chromatid, generating a 3:1 conversion. For this type of event, the transition between heterozygous and homozygous SNP markers occurs at different positions in the two sectors. The region outlined in blue defines the conversion tract. (C) Cross-over associated with repair of two sister chromatids to produce a 4:0 conversion tract. A DSB occurs on an unreplicated chromosome to produce two sister chromatids broken at the same position. The subsequent repair results in two conversion tracts of similar size, and one of the conversion events is associated with a cross-over. (D) Cross-over associated with a hybrid 3:1/4:0 conversion tract. The event is similar to that described for C, except the conversion tract associated with the repair of one break is longer than that associated with the second. (E) BIR event associated with a break in one chromatid. The net result of this event is a pink/white sector instead of a red/white sector.

Fig. 6.

Repair of DNA breaks induced in G1 or in S/G2 in wild-type strains and in strains with low levels of DNA polymerase-α. The widths of the arrows reflect the frequency of use of various pathways. (A) Spontaneous LOH events in wild-type cells. We suggest that most DSBs are formed in S or G2 (Right side of panel). The majority of these breaks are repaired by sister-chromatid recombination with the remainder being repaired using the homolog as a template. All of the G1-induced events involve exchange between the homologs. (B) LOH events induced by low levels of DNA polymerase-α. In these strains, the number of DSBs occurring in S is greatly elevated without an elevation in G1-initiated breaks. Consequently, the frequency of 3:1 events relative to 4:0 events is greatly elevated.