Recombination between homologous chromosomes induced by unrepaired UV-generated DNA damage requires Mus81p and is suppressed by Mms2p

Abstract

DNA lesions caused by UV radiation are highly recombinogenic. In wild-type cells, the recombinogenic effect of UV partially reflects the processing of UV-induced pyrimidine dimers into DNA gaps or breaks by the enzymes of the nucleotide excision repair (NER) pathway. In this study, we show that unprocessed pyrimidine dimers also potently induce recombination between homologs. In NER-deficient rad14 diploid strains, we demonstrate that unexcised pyrimidine dimers stimulate crossovers, noncrossovers, and break-induced replication events. The same dose of UV is about six-fold more recombinogenic in a repair-deficient strain than in a repair-proficient strain. We also examined the roles of several genes involved in the processing of UV-induced damage in NER-deficient cells. We found that the resolvase Mus81p is required for most of the UV-induced inter-homolog recombination events. This requirement likely reflects the Mus81p-associated cleavage of dimer-blocked replication forks. The error-free post-replication repair pathway mediated by Mms2p suppresses dimer-induced recombination between homologs, possibly by channeling replication-blocking lesions into recombination between sister chromatids.

Fig 1. Pathways of homologous recombination.

We depict recombination events initiated by a double-stranded DNA break (DSB) with each chromosome shown as a double-stranded DNA molecule. All pathways are initiated by the invasion of the 3’ end of the broken DNA molecule. Dotted lines denote DNA synthesis primed by the invading end. Regions in which a red strand and black strand are paired (in blue boxes) represent heteroduplexes; repair of mismatches within the heteroduplexes can generate gene conversion events. In synthesis-dependent strand annealing (SDSA), following DNA synthesis primed from the invading strand, the invading strand is displaced, hybridizing to the other broken end. This pathway produces a gene conversion that is not associated with a crossover (NCO). In Holliday junction (HJ) containing intermediates, an association of both broken ends with the intact template molecule can result in formation of a single Holliday junction (sHJ), a nicked Holliday junction, or a double Holliday junction (dHJ). Cleavage of these junctions by resolvases can produce either non-crossovers (NCO) or crossovers (CO). For both CO and NCO products, both molecules contain heteroduplexes located in trans. In contrast, if the HJ is resolved by dissolution, the heteroduplex regions are located in cis. In break-induced replication (BIR) events, the invading end copies the intact homolog by conservative DNA replication, resulting in a large terminal LOH event.

Fig 2. Patterns of loss of heterozygosity (LOH).

As described in the text, diploids were derived from two sequence-diverged haploids. The diploids were homozygous for the ochre suppressible ade2–1 mutation and heterozygous for the ochre suppressor SUP4-o tRNA (shown as a triangle) near the left end of chromosome V or the right end of IV. Zero, one, and two copies of SUP4-o in the diploid produce red, pink or white colonies, respectively. The black and red lines indicate the homolog derived from the haploids YJM789 and W303–1A, respectively. The boxed chromosomes on the right side of the Fig represent recombinant products. Next to these products, we show the patterns of heterozygous markers and homozygous markers (identified by SNP arrays) as lines. Chromosome regions that are heterozygous, homozygous for W303–1A-derived SNPs, or homozygous for YJM789-derived SNPs are represented by green, red, and black segments, respectively. D1 and D2 indicate the LOH patterns in the two daughter cells that contain the recombinant products. A. Simple crossover. When the red and white sectors are examined by SNP arrays, for this type of recombination event, D1 and D2 are identical in the positions of the transition between heterozygous and homozygous SNPs. This result is expected for a crossover without an associated gene conversion. B. Crossover with a 3:1 conversion tract. In this example, when the red and white sectors are examined, the transition positions between heterozygous and homozygous SNPs are not identical. In the region that is boxed in blue, considering both sectors, three of the chromosomes have “red” SNPs and only one has “black” SNPs. C. Crossover with a 4:0 conversion. In this red/white sectored colony, there is a region adjacent to the crossover in which all four chromosomes have the “red” SNP. We interpret this pattern as resulting from the repair of two sister chromatids that are broken at the same position. It is likely that these events are a consequence of a DSB in an unreplicated chromosome that is subsequently replicated. Repair of one of the broken chromatids is associated with a conversion and a crossover, whereas the second chromatid is repaired by a conversion event unassociated with a crossover. D. BIR event. A DSB on the black chromatid is repaired by a BIR event that duplicates sequences from the red chromatid. Note that this event can be detected on any of the chromosomes, not just the one marked with the SUP4-o gene. E. Conversion event unassociated with a crossover. As noted in Fig. 2D, because of the high frequency of LOH events in UV-treated cells we can detect classes A to E as unselected events.

Fig 3. Examples of LOH events as detected by SNP microarrays.

Cells were treated with UV and allowed to form colonies. We isolated DNA from the red and white sectors of sectored colonies, and examined the samples using SNP microarrays. Although these sectors were selected to have a reciprocal crossover on chromosome V, the two events shown below are unselected events on other chromosomes. Experimental samples were hybridized in competition with a differentially-labeled heterozygous control sample. On the Y-axis is shown the relative hybridization level of the experimental and control samples to SNP-specific oligonucleotides. Red and black colors indicate hybridization to W303–1A-specific and YJM789-specific SNPs, respectively. A value of 1 indicates heterozygosity. The X-axis shows SGD coordinates for the individual chromosomes. A. Unselected crossover on chromosome VIII. On chromosome VIII, there is a transition to homozygosity of red SNPs in the red sector and to homozygosity of black SNPs in the white sector. The positions of the transition are not identical in the sectors, indicating a 3:1 gene conversion (blue box) associated with the crossover. B. Unselected gene conversion event on chromosome II. In the red sector, there is a small interstitial LOH region. The white sector has no LOH events. This pattern would be produced by a gene conversion event unassociated with a crossover, or by a conversion event associated with a crossover in which the two recombinant chromosomes co-segregate.

Fig 4. Histogram of numbers of unselected LOH events per colony induced by UV.

Using SNP microarrays, we analyzed more than 20 colonies of each of three strains (rad14, rad14 mus81, and rad14 mms2) for unselected LOH events induced by 1 J/m². The numbers of LOH per colony are shown in this figure. The color code is: rad14 (blue), rad14 mus81 (pink), and rad14 mms2 (purple). The dotted lines indicate the average number of LOH events per single colony using the same color code.

Fig 5. Recombination events induced by persistent UV damage in NER-deficient yeast strains.

UV treatment results in the formation of a pyrimidine dimer (circle). A. Generation of a recombinogenic DSB. We suggest that the dimer-associated replication fork block can be converted into a DSB by the structure-nuclease Mus81p. The resulting DSB can be repaired either by recombination with the homolog, resulting in a detectable LOH event (boxed in grey), or by recombination with the sister chromatid which does not lead to LOH. B. Error-free bypass of lesions. When the 3’ end on the leading strand encounters a dimer, it can invade the sister chromatid (template switch), and continue replication. This branch of the post-replication repair requires Mms2p. We think that this pathway mainly involves interaction between sister chromatids without causing LOH and suppresses recombination between homologs.