Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

Yin, Yi; Petes, Thomas D.

doi:10.1371/journal.pgen.1003894

Cited by 61 publications

(128 citation statements)

References 58 publications

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“…In this depiction, red and blue lines represent levels of hybridization to W303-1A-and YJM789-derived SNPs, respectively. An interstitial LOH event is shown in Figure 1A; from previous studies Yin and Petes 2013), we know that these events reflect gene conversion, the nonreciprocal transfer of sequences from one homolog to the other. In this example, sequences of the YJM789 copy of chromosome II were copied into the W303-1A copy of chromosome II at two different locations.…”

Section: Resultsmentioning

confidence: 87%

“…The transition between heterozygous and homozygous SNPs should contain the site of the recombination-initiating lesion Yin and Petes 2013). Breakpoint regions in subcultured mutant strains were examined to determine if specific chromosome elements (for example, autonomously replicating sequence, ARS elements) were overrepresented at the breakpoints.…”

Section: Association Of Genome Features With Recombination Eventsmentioning

confidence: 99%

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Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity

O’Connell¹,

Jinks-Robertson

Petes

2015

Genetics

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Two types of RNA:DNA associations can lead to genome instability: the formation of R-loops during transcription and the incorporation of ribonucleotide monophosphates (rNMPs) into DNA during replication. Both ribonuclease (RNase) H1 and RNase H2 degrade the RNA component of R-loops, whereas only RNase H2 can remove one or a few rNMPs from DNA. We performed highresolution mapping of mitotic recombination events throughout the yeast genome in diploid strains of Saccharomyces cerevisiae lacking RNase H1 (rnh1D), RNase H2 (rnh201D), or both RNase H1 and RNase H2 (rnh1D rnh201D). We found little effect on recombination in the rnh1D strain, but elevated recombination in both the rnh201D and the double-mutant strains; levels of recombination in the double mutant were 50% higher than in the rnh201 single-mutant strain. An rnh201D mutant that additionally contained a mutation that reduces rNMP incorporation by DNA polymerase e (pol2-M644L) had a level of instability similar to that observed in the presence of wild-type Pol e. This result suggests that the elevated recombination observed in the absence of only RNase H2 is primarily a consequence of R-loops rather than misincorporated rNMPs.KEYWORDS RNase H1; RNase H2; loss of heterozygosity; mitotic recombination; microarrays R NA transcripts can stably associate with the DNA template during transcription, giving rise to R-loop structures containing an RNA:DNA hybrid and an unpaired DNA strand. Such R-loops can stall DNA replication forks and are a major source of recombination events that are stimulated by high levels of transcription (reviewed by Aguilera and Garcia-Muse 2012). Transcription-associated recombination (TAR) is generally strongest when the transcription machinery and a replication fork approach each other head on (Prado and Aguilera 2005), but there is also an orientation-independent component to such conflicts ). Importantly, TAR is highly elevated when RNA processing is perturbed and R-loops accumulate, or when R-loop removal mechanisms are disabled. The RNA component of R-loops can be removed by the Sen1 RNA-DNA helicase (Mischo et al. 2011) or degraded by ribonuclease (RNase) H1 or RNase H2 (reviewed in Cerritelli and Crouch 2009), which are generally considered to be functionally redundant.R-loops in yeast have been quantified genome-wide using immunoprecipitation with an antibody specific to DNA:RNA hybrids, followed by hybridization to a microarray or by DNA sequencing. This type of analysis has been done in wild type (Chan et al. 2014;El Hage et al. 2014), mRNA processing defective (Chan et al. 2014), RNase H-defective (Chan et al. 2014;El Hage et al. 2014), and RNA-DNA helicase (Sen1)-defective strains (Chan et al. 2014). In wild-type strains, Chan et al. (2014) observed RNA-DNA hybrid accumulation at Ty retrotransposons, near telomeres, within the ribosomal RNA (rRNA) gene cluster, and at highly transcribed GC-rich genes. El Hage et al. (2014) found hybrid accumulation in wild-type strains at highly transcribed genes, within the rRNA gen...

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Section: Resultsmentioning

confidence: 87%

Section: Association Of Genome Features With Recombination Eventsmentioning

confidence: 99%

Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity

O’Connell¹,

Jinks-Robertson

Petes

2015

Genetics

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“…Since mitotic crossover can result in LOH throughout the region centromere-distal to the crossover, which can be detrimental, it seems logical that noncrossover repair pathways would predominate. This hypothesis has been supported by several studies that found crossover-associated conversions to be approximately one-quarter to one-third of all conversions (Haber and Hearn 1985;Chua and Jinks-Robertson 1991;Inbar and Kupiec 1999;Nickoloff et al 1999;Yin and Petes 2013), but three studies reported that half or nearly half of all conversions were crossover-associated (Aguilera and Klein 1989;Welz-Voegele and Jinks-Robertson 2008;Yim et al 2014). In this study, among the 79 gene conversions collected from cells grown under replication stress (no galactose), 30% were crossover-associated and 70% were noncrossoverassociated.…”

mentioning

confidence: 76%

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

Chumki¹,

Dunn²,

Coates³

et al. 2016

Genetics

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Replication stress causes breaks at chromosomal locations called common fragile sites. Deletions causing loss of heterozygosity (LOH) in human tumors are strongly correlated with common fragile sites, but the role of gene conversion in LOH at fragile sites in tumors is less well studied. Here, we investigated gene conversion stimulated by instability at fragile site FS2 in the yeast Saccharomyces cerevisiae. In our screening system, mitotic LOH events near FS2 are identified by production of red/white sectored colonies. We analyzed single nucleotide polymorphisms between homologs to determine the cause and extent of LOH. Instability at FS2 increases gene conversion 48-to 62-fold, and conversions unassociated with crossover represent 6-7% of LOH events. Gene conversion can result from repair of mismatches in heteroduplex DNA during synthesis-dependent strand annealing (SDSA), double-strand break repair (DSBR), and from break-induced replication (BIR) that switches templates [double BIR (dBIR)]. It has been proposed that SDSA and DSBR typically result in shorter gene-conversion tracts than dBIR. In cells under replication stress, we found that bidirectional tracts at FS2 have a median length of 40.8 kb and a wide distribution of lengths; most of these tracts are not crossover-associated. Tracts that begin at the fragile site FS2 and extend only distally are significantly shorter. The high abundance and long length of noncrossover, bidirectional gene-conversion tracts suggests that dBIR is a prominent mechanism for repair of lesions at FS2, thus this mechanism is likely to be a driver of common fragile site-stimulated LOH in human tumors.KEYWORDS loss of heterozygosity; gene conversion; BIR; fragile site; homologous recombination D NA integrity is frequently challenged by chemical, environmental, and physical agents, as well as by endogenous factors. Common fragile sites are one source of endogenous damage, causing DNA instability and breaks under conditions of replication stress when DNA replication is partially inhibited (Durkin and Glover 2007;Sarni and Kerem 2016). Replication stress has typically been induced experimentally by treating cells with the polymerase inhibitor aphidicolin (Glover et al. 1984) or by genetically lowering the levels of DNA polymerases Lemoine et al. 2008). In vivo, replication stress has been observed early in the process of tumor development as a result of alteration of replication fork progression and nucleotide deficiency caused by activation of oncogenes (Di Micco et al. 2006;Bester et al. 2011). In cancer cells, human common fragile sites are hotspots for chromosomal deletions, amplifications, and rearrangements (Burrow et al. 2009;Drusco et al. 2011; OzeriGalai et al. 2011 OzeriGalai et al. , 2012, and a large-scale screen of deletions in 746 cancer cell lines reported that many areas of unexplained deletions are in fragile sites (Bignell et al. 2010).The alterations at human common fragile sites in cancer cells are proposed to result from the processes of DNA repair at...

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“…In most previous studies, the mechanics of the assay system limited the extent of the geneconversion event to 2 kb or less. It is not clear, therefore, whether long conversion tracts observed in our study are generated solely through this mechanism or may involve alternative pathways, such as repair of a double-stranded DNA gap or an interstitial BIR event that switches templates (23).…”

Section: Analysis Of Conversion Tracts Associated With Reciprocal Cromentioning

confidence: 91%

“…The experiments described above did not allow us to distinguish cross-overs and BIR events, nor to examine the types of gene conversions associated with cross-overs. For this analysis, we used diploid strains in which cross-overs could be detected by a sectoring assay (22,23,26). In these strains, one homolog has the ochre-suppressor SUP4-o located near the telomere and the hygromycin-resistant HYG gene located at a similar position on the other homolog; in addition, the diploid is homozygous for ade2-1, an ochre mutation that, when unsuppressed, results in red colonies.…”

Section: Experimental Approach For Mapping Regions Of Loss Of Heterozmentioning

confidence: 99%

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

Song

Dominska

Greenwell

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

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107

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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.mitotic cross-overs | loss of heterozygosity | break-induced replication

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Genome-Wide High-Resolution Mapping of UV-Induced Mitotic Recombination Events in Saccharomyces cerevisiae

Cited by 61 publications

References 58 publications

Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity

Elevated Genome-Wide Instability in Yeast Mutants Lacking RNase H Activity

Remarkably Long-Tract Gene Conversion Induced by Fragile Site Instability inSaccharomyces cerevisiae

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

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