Sequence Divergence Impedes Crossover More Than Noncrossover Events During Mitotic Gap Repair in Yeast

Welz-Voegele, Caroline; Jinks-Robertson, Sue

doi:10.1534/genetics.108.090233

Cited by 37 publications

(56 citation statements)

References 67 publications

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“…In yeast, sgs1⌬ mutants demonstrate a hyperrecombination phenotype when spontaneous homologous recombination is analyzed (22,65,68), but HO-induced recombination (28) and plasmid gap repair (66) are slightly decreased, consistent with our results. In Drosophila, BLM deficiency is associated with a marked decrease in SDSA during gap repair (1,36), which has been proposed to be related to a requirement for helicase activity to support multiple rounds of strand invasion.…”

Section: Discussionsupporting

confidence: 90%

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

LaRocque

Jasin

2010

Molecular and Cellular Biology

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Double-strand breaks (DSBs) are particularly deleterious DNA lesions for which cells have developed multiple mechanisms of repair. One major mechanism of DSB repair in mammalian cells is homologous recombination (HR), whereby a homologous donor sequence is used as a template for repair. For this reason, HR repair of DSBs is also being exploited for gene modification in possible therapeutic approaches. HR is sensitive to sequence divergence, such that the cell has developed ways to suppress recombination between diverged ("homeologous") sequences. In this report, we have examined several aspects of HR between homeologous sequences in mouse and human cells. We found that gene conversion tracts are similar for mouse and human cells and are generally <100 bp, even in Msh2 ؊/؊ cells which fail to suppress homeologous recombination. Gene conversion tracts are mostly unidirectional, with no observed mutations. Additionally, no alterations were observed in the donor sequences. While both mouse and human cells suppress homeologous recombination, the suppression is substantially less in the transformed human cells, despite similarities in the gene conversion tracts. BLM-deficient mouse and human cells suppress homeologous recombination to a similar extent as wild-type cells, unlike Sgs1-deficient Saccharomyces cerevisiae.The ability of a cell to repair DNA damage is integral to maintaining genome integrity. One common type of damage that is particularly detrimental is a double-strand break (DSB), where both strands of DNA are broken. If not accurately repaired, DSBs can lead to cell death, chromosomal rearrangements, and loss of genetic material (reviewed in references 14 and 19). One mechanism of DSB repair is homologous recombination (HR), in which an unbroken homologous sequence, the donor of genetic information, is used as a template for repair of the broken sequence, the recipient of genetic information. HR intermediates possess heteroduplex DNA (hDNA), where one strand of DNA is derived from the donor sequence, and the second strand is derived from the recipient sequence. Mismatches in hDNA are substrates of the mismatch repair machinery (MMR) (reviewed in reference 38), leading to gene conversion. HR is the preferred repair pathway of DSBs in Saccharomyces cerevisiae (reviewed in references 42 and 46), plays an important role in repair of DSBs in Drosophila (1, 32), and is a major repair pathway of DSBs that occur during S/G 2 in mammalian cells (33,54).Two pathways appear to predominate for the repair of DSBs by HR, both of which can give rise to noncrossover products, which predominate in mitotic mammalian cells (Fig. 1) (29,52,60). In the DSB repair model proposed by Szostak et al. (61), double Holliday junctions are resolved to result in recombinant products (Fig. 1A). More recent evidence suggests the existence of an alternative pathway, termed synthesis-dependent strand annealing (SDSA) (Fig. 1B) (20,40,42,52). One difference between these two pathways is that the DSB repair model requires capture of both DNA...

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Section: Discussionsupporting

confidence: 90%

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

LaRocque

Jasin

2010

Molecular and Cellular Biology

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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.…”

Section: Association Of Mitotic Gene-conversion Tracts At Fs2 With Crmentioning

confidence: 83%

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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“…CO suppression is supported biochemically because BLM, together with TOPIIIα and RMI1, migrates Holliday junctions to promote their dissolution (14). Genetic evidence in both yeast and Drosophila support a role for the respective BLM homologs (Sgs1 and MUS309, respectively) in suppressing interhomolog COs at DNA lesions (15)(16)(17). A recent study in human cells is consistent with a similar role of mammalian Blm, although crossing over could not be distinguished from other repair pathways (18).…”

mentioning

confidence: 89%

Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells

LaRocque

Stark

et al. 2011

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

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Genomic integrity often is compromised in tumor cells, as illustrated by genetic alterations leading to loss of heterozygosity (LOH). One mechanism of LOH is mitotic crossover recombination between homologous chromosomes, potentially initiated by a double-strand break (DSB). To examine LOH associated with DSBinduced interhomolog recombination, we analyzed recombination events using a reporter in mouse embryonic stem cells derived from F1 hybrid embryos. In this study, we were able to identify LOH events although they occur only rarely in wild-type cells (≤2.5%). The low frequency of LOH during interhomolog recombination suggests that crossing over is rare in wild-type cells. Candidate factors that may suppress crossovers include the RecQ helicase deficient in Bloom syndrome cells (BLM), which is part of a complex that dissolves recombination intermediates. We analyzed interhomolog recombination in BLM-deficient cells and found that, although interhomolog recombination is slightly decreased in the absence of BLM, LOH is increased by fivefold or more, implying significantly increased interhomolog crossing over. These events frequently are associated with a second homologous recombination event, which may be related to the mitotic bivalent structure and/or the cell-cycle stage at which the initiating DSB occurs.double-strand break repair | gene conversion L oss of heterozygosity (LOH) is a major mechanism for uncovering mutations in tumor-suppressor genes. Examination of tumor cells and model studies in cell lines have established several mechanisms of LOH, including chromosome loss/duplication (uniparental disomy), mitotic recombination between homologous chromosomes, and gross chromosomal rearrangement as a result of genome-wide chromosomal instability (1-4). LOH caused by homologous recombination (HR) can arise from crossing over between homologous chromosomes, followed by segregation of crossover (CO) chromatids to separate daughter cells (CO-LOH). In this way, the entire chromosome arm distal to the point of crossing over is homozygozed for genetic information. This process contrasts with interhomolog noncrossover (NCO) events during which gene conversion gives rise to only small regions of LOH (5). Chromosomal double-strand breaks (DSBs) may be initiating lesions for CO-LOH, because they induce interhomolog crossing over in meiotic cells (reviewed in ref. 6). Although DSB-induced crossing over is clearly necessary during the specialized first meiotic division (6), a role for interhomolog crossing over in mitotic cells is uncertain.DSBs induce mitotic interhomolog HR in mammalian cells (7), but CO-LOH occurs infrequently during DSB repair (5, 8), suggesting that factors actively suppress crossing over. Candidate factors include the RecQ helicase deficient in cells from Bloom syndrome patients (BLM) (9). Consistent with this notion, levels of spontaneous intersister COs, i.e., sister-chromatid exchanges, are elevated in both human and mouse BLM-deficient cells (10, 11), as is the frequency of spontaneous LOH (9...

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Sequence Divergence Impedes Crossover More Than Noncrossover Events During Mitotic Gap Repair in Yeast

Cited by 37 publications

References 67 publications

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

Mechanisms of Recombination between Diverged Sequences in Wild-Type and BLM-Deficient Mouse and Human Cells

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

Interhomolog recombination and loss of heterozygosity in wild-type and Bloom syndrome helicase (BLM)-deficient mammalian cells

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