Mechanisms underlying genome instability mediated by formation of foldback inversions in Saccharomyces cerevisiae

Foldback inversions, also called inverted duplications, have been observed in human genetic diseases and cancers. Here, we used a Saccharomyces cerevisiae genetic system that generates gross chromosomal rearrangements (GCRs) mediated by foldback inversions combined with whole-genome sequencing to study their formation. Foldback inversions were mediated by formation of single-stranded DNA hairpins. Two types of hairpins were identified: small-loop hairpins that were suppressed by MRE11, SAE2, SLX1, and YKU80 and large-loop hairpins that were suppressed by YEN1, TEL1, SWR1, and MRC1. Analysis of CRISPR/Cas9-induced double strand breaks (DSBs) revealed that long-stem hairpin-forming sequences could form foldback inversions when proximal or distal to the DSB, whereas short-stem hairpin-forming sequences formed foldback inversions when proximal to the DSB. Finally, we found that foldback inversion GCRs were stabilized by secondary rearrangements, mostly mediated by different homologous recombination mechanisms including single-strand annealing; however, POL32-dependent break-induced replication did not appear to be involved forming secondary rearrangements.

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Genetic Steps of Mammalian Homologous Repair with Distinct Mutagenic Consequences

Molecular and Cellular Biology ◽

10.1128/mcb.24.21.9305-9316.2004 ◽

2004 ◽

Vol 24 (21) ◽

pp. 9305-9316 ◽

Cited By ~ 313

Author(s):

Jeremy M. Stark ◽

Andrew J. Pierce ◽

Jin Oh ◽

Albert Pastink ◽

Maria Jasin

Keyword(s):

Mammalian Cells ◽

Atp Hydrolysis ◽

Chromosomal Rearrangements ◽

Strand Break ◽

Dsb Repair ◽

Mutagenic Potential ◽

Gross Chromosomal Rearrangements ◽

Single Strand Annealing ◽

Chromosomal Breaks ◽

Strand Annealing

ABSTRACT Repair of chromosomal breaks is essential for cellular viability, but misrepair generates mutations and gross chromosomal rearrangements. We investigated the interrelationship between two homologous-repair pathways, i.e., mutagenic single-strand annealing (SSA) and precise homology-directed repair (HDR). For this, we analyzed the efficiency of repair in mammalian cells in which double-strand break (DSB) repair components were disrupted. We observed an inverse relationship between HDR and SSA when RAD51 or BRCA2 was impaired, i.e., HDR was reduced but SSA was increased. In particular, expression of an ATP-binding mutant of RAD51 led to a >90-fold shift to mutagenic SSA repair. Additionally, we found that expression of an ATP hydrolysis mutant of RAD51 resulted in more extensive gene conversion, which increases genetic loss during HDR. Disruption of two other DSB repair components affected both SSA and HDR, but in opposite directions: SSA and HDR were reduced by mutation of Brca1, which, like Brca2, predisposes to breast cancer, whereas SSA and HDR were increased by Ku70 mutation, which affects nonhomologous end joining. Disruption of the BRCA1-associated protein BARD1 had effects similar to those of mutation of BRCA1. Thus, BRCA1/BARD1 has a role in homologous repair before the branch point of HDR and SSA. Interestingly, we found that Ku70 mutation partially suppresses the homologous-repair defects of BARD1 disruption. We also examined the role of RAD52 in homologous repair. In contrast to yeast, Rad52 − / − mouse cells had no detectable HDR defect, although SSA was decreased. These results imply that the proper genetic interplay of repair factors is essential to limit the mutagenic potential of DSB repair.

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Inverted DNA Repeats Channel Repair of Distant Double-Strand Breaks into Chromatid Fusions and Chromosomal Rearrangements

Molecular and Cellular Biology ◽

10.1128/mcb.01740-06 ◽

2007 ◽

Vol 27 (7) ◽

pp. 2601-2614 ◽

Cited By ~ 70

Author(s):

Kelly VanHulle ◽

Francene J. Lemoine ◽

Vidhya Narayanan ◽

Brandon Downing ◽

Krista Hull ◽

...

Keyword(s):

Chromosomal Rearrangements ◽

Inverted Repeats ◽

Dna Repeats ◽

Dna Breaks ◽

Strand Breaks ◽

Gross Chromosomal Rearrangements ◽

Double Strand Dna Breaks ◽

Single Strand Annealing ◽

Strand Annealing ◽

Break Induced Replication

ABSTRACT Inverted DNA repeats are known to cause genomic instabilities. Here we demonstrate that double-strand DNA breaks (DSBs) introduced a large distance from inverted repeats in the yeast (Saccharomyces cerevisiae) chromosome lead to a burst of genomic instability. Inverted repeats located as far as 21 kb from each other caused chromosome rearrangements in response to a single DSB. We demonstrate that the DSB initiates a pairing interaction between inverted repeats, resulting in the formation of large dicentric inverted dimers. Furthermore, we observed that propagation of cells containing inverted dimers led to gross chromosomal rearrangements, including translocations, truncations, and amplifications. Finally, our data suggest that break-induced replication is responsible for the formation of translocations resulting from anaphase breakage of inverted dimers. We propose a model explaining the formation of inverted dicentric dimers by intermolecular single-strand annealing (SSA) between inverted DNA repeats. According to this model, anaphase breakage of inverted dicentric dimers leads to gross chromosomal rearrangements (GCR). This “SSA-GCR” pathway is likely to be important in the repair of isochromatid breaks resulting from collapsed replication forks, certain types of radiation, or telomere aberrations that mimic isochromatid breaks.

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Functions of Saccharomyces cerevisiae 14-3-3 Proteins in Response to DNA Damage and to DNA Replication Stress

Genetics ◽

10.1093/genetics/165.4.1717 ◽

2003 ◽

Vol 165 (4) ◽

pp. 1717-1732

Author(s):

Francisca Lottersberger ◽

Fabio Rubert ◽

Veronica Baldo ◽

Giovanna Lucchini ◽

Maria Pia Longhese

Keyword(s):

Saccharomyces Cerevisiae ◽

Dna Damage ◽

Dna Replication ◽

Genome Instability ◽

Chromosomal Rearrangements ◽

Replication Stress ◽

Checkpoint Kinases ◽

Gross Chromosomal Rearrangements ◽

Dna Damage Checkpoints ◽

Dna Replication Stress

Abstract Two members of the 14-3-3 protein family, involved in key biological processes in different eukaryotes, are encoded by the functionally redundant Saccharomyces cerevisiae BMH1 and BMH2 genes. We produced and characterized 12 independent bmh1 mutant alleles, whose presence in the cell as the sole 14-3-3 source causes hypersensitivity to genotoxic agents, indicating that Bmh proteins are required for proper response to DNA damage. In particular, the bmh1-103 and bmh1-266 mutant alleles cause defects in G1/S and G2/M DNA damage checkpoints, whereas only the G2/M checkpoint is altered by the bmh1-169 and bmh1-221 alleles. Impaired checkpoint responses correlate with the inability to maintain phosphorylated forms of Rad53 and/or Chk1, suggesting that Bmh proteins might regulate phosphorylation/dephosphorylation of these checkpoint kinases. Moreover, several bmh1 bmh2Δ mutants are defective in resuming DNA replication after transient deoxynucleotide depletion, and all display synthetic effects when also carrying mutations affecting the polα-primase and RPA DNA replication complexes, suggesting a role for Bmh proteins in DNA replication stress response. Finally, the bmh1-169 bmh2Δ and bmh1-170 bmh2Δ mutants show increased rates of spontaneous gross chromosomal rearrangements, indicating that Bmh proteins are required to suppress genome instability.

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DNA replication machinery prevents Rad52-dependent single-strand annealing that leads to gross chromosomal rearrangements at centromeres

Communications Biology ◽

10.1038/s42003-020-0934-0 ◽

2020 ◽

Vol 3 (1) ◽

Cited By ~ 1

Author(s):

Atsushi T. Onaka ◽

Jie Su ◽

Yasuhiro Katahira ◽

Crystal Tang ◽

Faria Zafar ◽

...

Keyword(s):

Dna Replication ◽

Chromosomal Rearrangements ◽

Repetitive Sequences ◽

Single Strand ◽

Replication Machinery ◽

Gross Chromosomal Rearrangements ◽

Single Strand Annealing ◽

Rad52 Protein ◽

Strand Annealing ◽

Recombination Pathway

AbstractHomologous recombination between repetitive sequences can lead to gross chromosomal rearrangements (GCRs). At fission yeast centromeres, Rad51-dependent conservative recombination predominantly occurs between inverted repeats, thereby suppressing formation of isochromosomes whose arms are mirror images. However, it is unclear how GCRs occur in the absence of Rad51 and how GCRs are prevented at centromeres. Here, we show that homology-mediated GCRs occur through Rad52-dependent single-strand annealing (SSA). The rad52-R45K mutation, which impairs SSA activity of Rad52 protein, dramatically reduces isochromosome formation in rad51 deletion cells. A ring-like complex Msh2–Msh3 and a structure-specific endonuclease Mus81 function in the Rad52-dependent GCR pathway. Remarkably, mutations in replication fork components, including DNA polymerase α and Swi1/Tof1/Timeless, change the balance between Rad51-dependent recombination and Rad52-dependent SSA at centromeres, increasing Rad52-dependent SSA that forms isochromosomes. Our results uncover a role of DNA replication machinery in the recombination pathway choice that prevents Rad52-dependent GCRs at centromeres.

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Essential Saccharomyces cerevisiae genome instability suppressing genes identify potential human tumor suppressors

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906921116 ◽

2019 ◽

Vol 116 (35) ◽

pp. 17377-17382 ◽

Cited By ~ 2

Author(s):

Anjana Srivatsan ◽

Binzhong Li ◽

Dafne N. Sanchez ◽

Steven B. Somach ◽

Vandeclecio L. da Silva ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Genome Instability ◽

Protein Complexes ◽

Chromosomal Rearrangements ◽

Human Tumor ◽

Temperature Sensitive ◽

Temperature Sensitive Mutants ◽

Gross Chromosomal Rearrangements ◽

Human Genes ◽

Significant Enrichment

Gross Chromosomal Rearrangements (GCRs) play an important role in human diseases, including cancer. Although most of the nonessential Genome Instability Suppressing (GIS) genes in Saccharomyces cerevisiae are known, the essential genes in which mutations can cause increased GCR rates are not well understood. Here 2 S. cerevisiae GCR assays were used to screen a targeted collection of temperature-sensitive mutants to identify mutations that caused increased GCR rates. This identified 94 essential GIS (eGIS) genes in which mutations cause increased GCR rates and 38 candidate eGIS genes that encode eGIS1 protein-interacting or family member proteins. Analysis of TCGA data using the human genes predicted to encode the proteins and protein complexes implicated by the S. cerevisiae eGIS genes revealed a significant enrichment of mutations affecting predicted human eGIS genes in 10 of the 16 cancers analyzed.

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Mitotic checkpoint function in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.0407010101 ◽

2004 ◽

Vol 101 (45) ◽

pp. 15980-15985 ◽

Cited By ~ 37

Author(s):

K. Myung ◽

S. Smith ◽

R. D. Kolodner

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosomal Rearrangements ◽

Mitotic Checkpoint ◽

Gross Chromosomal Rearrangements

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Suppression of gross chromosomal rearrangements by the multiple functions of the Mre11–Rad50–Xrs2 complex in Saccharomyces cerevisiae

DNA Repair ◽

10.1016/j.dnarep.2005.01.004 ◽

2005 ◽

Vol 4 (5) ◽

pp. 606-617 ◽

Cited By ~ 26

Author(s):

Stephanie Smith ◽

Amitabha Gupta ◽

Richard D. Kolodner ◽

Kyungjae Myung

Keyword(s):

Saccharomyces Cerevisiae ◽

Chromosomal Rearrangements ◽

Multiple Functions ◽

Gross Chromosomal Rearrangements

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Meiosis-specific recombinase Dmc1 is a potent inhibitor of the Srs2 antirecombinase

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1810457115 ◽

2018 ◽

Vol 115 (43) ◽

pp. E10041-E10048 ◽

Cited By ~ 14

Author(s):

J. Brooks Crickard ◽

Kyle Kaniecki ◽

Youngho Kwon ◽

Patrick Sung ◽

Eric C. Greene

Keyword(s):

Chromosome Segregation ◽

Single Molecule ◽

Atp Hydrolysis ◽

Potent Inhibitor ◽

Chromosomal Rearrangements ◽

Motor Protein ◽

Product Formation ◽

Gross Chromosomal Rearrangements ◽

Strand Annealing ◽

Hydrolysis Activity

Cross-over recombination products are a hallmark of meiosis because they are necessary for accurate chromosome segregation and they also allow for increased genetic diversity during sexual reproduction. However, cross-overs can also cause gross chromosomal rearrangements and are therefore normally down-regulated during mitotic growth. The mechanisms that enhance cross-over product formation upon entry into meiosis remain poorly understood. In Saccharomyces cerevisiae, the Superfamily 1 (Sf1) helicase Srs2, which is an ATP hydrolysis-dependent motor protein that actively dismantles recombination intermediates, promotes synthesis-dependent strand annealing, the result of which is a reduction in cross-over recombination products. Here, we show that the meiosis-specific recombinase Dmc1 is a potent inhibitor of Srs2. Biochemical and single-molecule assays demonstrate that Dmc1 acts by inhibiting Srs2 ATP hydrolysis activity, which prevents the motor protein from undergoing ATP hydrolysis-dependent translocation on Dmc1-bound recombination intermediates. We propose a model in which Dmc1 helps contribute to cross-over formation during meiosis by antagonizing the antirecombinase activity of Srs2.

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