System to monitor crossovers on chromosome XII.

<p>A diploid was constructed with heterozygous markers immediately centromere-proximal to the ribosomal RNA (rRNA) gene clusters (<i>HYG</i>), within the rRNA gene cluster (<i>TRP1</i>), and immediately centromere-distal to the cluster (<i>URA3</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003894#pgen.1003894-Casper1" target="_blank">[34]</a>. G1-synchronized cells were treated with UV, plated on solid medium and grown non-selectively. The resulting colonies were replica-plated to medium lacking uracil, tryptophan, or containing hygromycin as described in the text. A. Sectoring pattern expected for a crossover centromere-proximal to the <i>HYG</i> marker. B. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-proximal to <i>TRP1</i>. C. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-distal to <i>TRP1</i>.</p

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

<p>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.</p

System to monitor crossovers on chromosome XII.

<p>A diploid was constructed with heterozygous markers immediately centromere-proximal to the ribosomal RNA (rRNA) gene clusters (<i>HYG</i>), within the rRNA gene cluster (<i>TRP1</i>), and immediately centromere-distal to the cluster (<i>URA3</i>) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003894#pgen.1003894-Casper1" target="_blank">[34]</a>. G1-synchronized cells were treated with UV, plated on solid medium and grown non-selectively. The resulting colonies were replica-plated to medium lacking uracil, tryptophan, or containing hygromycin as described in the text. A. Sectoring pattern expected for a crossover centromere-proximal to the <i>HYG</i> marker. B. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-proximal to <i>TRP1</i>. C. Sectoring pattern expected for a crossover within the rRNA gene cluster centromere-distal to <i>TRP1</i>.</p

Numbers of unselected UV-induced LOH events, deletions/duplications, and chromosome number changes in sectored colonies derived from UV-treated wild-type, <i>rad14</i>, <i>mus81</i>, and <i>yen1</i> diploids.¹

<p>¹For all strains, we examined <u>unselected</u> LOH events and other genomic changes in sectored colonies resulting from crossovers on chromosome V.</p><p>²We operationally define a BIR event as a terminal LOH event that is present in one sector but not the other. As discussed in the text, since we purify single colonies from each sector, we cannot exclude the possibility that some of the putative BIR events are actually crossovers that occur in the second division after irradiation.</p><p>³<u>p</u><0.001 compared to wild-type cells treated at 1 J/m².</p><p>⁴<u>p</u><0.01 compared to wild-type cells treated at 15 J/m².</p><p>⁵<u>p</u><0.01 compared to wild-type cells treated at 1 J/m².</p><p>Numbers of unselected UV-induced LOH events, deletions/duplications, and chromosome number changes in sectored colonies derived from UV-treated wild-type, <i>rad14</i>, <i>mus81</i>, and <i>yen1</i> diploids.<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005026#t002fn001" target="_blank">¹</a></p

Selected SCB and DSCB conversions in strains treated with 1/m² and 15 J/m².

<p>SCB and DSCB events are indicated in gray and red, respectively.</p

Numbers of unselected UV-induced LOH events and other genomic changes in single colonies derived from <i>rad14</i>, <i>rad14 mus81</i>, <i>rad14 yen1</i>, and <i>rad14 mms2</i> diploids treated with 1 J/m² of UV.

<p>¹One isolate also had a translocation.</p><p>²p<0.0001 compared to <i>rad14</i>.</p><p>³<u>p</u><0.001 compared to <i>rad14</i>.</p><p>⁴<u>p</u><0.01 compared to <i>rad14</i>.</p><p>Numbers of unselected UV-induced LOH events and other genomic changes in single colonies derived from <i>rad14</i>, <i>rad14 mus81</i>, <i>rad14 yen1</i>, and <i>rad14 mms2</i> diploids treated with 1 J/m² of UV.</p

Pathways of homologous recombination.

<p>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 <i>trans</i>. In contrast, if the HJ is resolved by dissolution, the heteroduplex regions are located in <i>cis</i>. In break-induced replication (BIR) events, the invading end copies the intact homolog by conservative DNA replication, resulting in a large terminal LOH event.</p

Recombination between Homologous Chromosomes Induced by Unrepaired UV-Generated DNA Damage Requires Mus81p and Is Suppressed by Mms2p

<div><p>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 <i>rad14</i> 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.</p></div

Patterns of loss of heterozygosity (LOH).

<p>As described in the text, diploids were derived from two sequence-diverged haploids. The diploids were homozygous for the ochre suppressible <i>ade2–1</i> mutation and heterozygous for the ochre suppressor <i>SUP4-o</i> tRNA (shown as a triangle) near the left end of chromosome V or the right end of IV. Zero, one, and two copies of <i>SUP4-o</i> 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 <i>SUP4-o</i> gene. E. Conversion event unassociated with a crossover. As noted in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005026#pgen.1005026.g002" target="_blank">Fig. 2D</a>, because of the high frequency of LOH events in UV-treated cells we can detect classes A to E as unselected events.</p

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

<p>Using SNP microarrays, we analyzed more than 20 colonies of each of three strains (r<i>ad14</i>, <i>rad14 mus81</i>, and <i>rad14 mms2</i>) for unselected LOH events induced by 1 J/m². The numbers of LOH per colony are shown in this figure. The color code is: <i>rad14</i> (blue), <i>rad14 mus81</i> (pink), and <i>rad14 mms2</i> (purple). The dotted lines indicate the average number of LOH events per single colony using the same color code.</p