Bridge-Induced Chromosome Translocation in Yeast Relies upon a Rad54/Rdh54-Dependent, Pol32-Independent Pathway

Tosato, Valentina; Sidari, Sabrina; Bruschi, Carlo V.

doi:10.1371/journal.pone.0060926

Cited by 9 publications

(24 citation statements)

References 64 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…While the rate of aneuploidy in RDH54+ diploids (0.90 × 10 -4 ) is very similar to previous observations from diploid yeast ((5); rate 1.04 × 10 -4 ), we found that rdh54Δ elevated the rate of aneuploidy 4-fold overall and 4.5-fold within diploids ( Fig. 2; binomial test: P < 10 -4 ), indicating that RDH54 plays a role in mitotic chromosome segregation in addition to its role in meiosis (8,11), as suggested previously (12). Given the absence of aneuploidy in haploids, we cannot determine whether the increase in aneuploidy in rdh54Δ lines also applies to haploids or is unique to diploids.…”

Section: Resultssupporting

confidence: 92%

The genome-wide rate and spectrum of spontaneous mutations differs between haploid and diploid yeast

Sharp

Sandell

James

et al. 2018

Preprint

View full text Add to dashboard Cite

By altering the dynamics of DNA replication and repair, alternative ploidy states may experience different rates and types of new mutations, leading to divergent evolutionary outcomes. We report the first direct comparison of the genome-wide spectrum of spontaneous mutations arising in haploid and diploid forms of the budding yeast Saccharomyces cerevisiae. Characterizing the number, types, locations, and effects of thousands of mutations revealed that haploids were more prone to single-nucleotide and mitochondrial mutations, while larger structural changes were more common in diploids. Mutations were more likely to be detrimental in diploids, even after accounting for the large impact of structural changes, contrary to the prediction that diploidy masks the effects of recessive alleles. Haploidy is expected to reduce the opportunity for conservative DNA repair involving homologous chromosomes, increasing the insertion-deletion rate, but we found little support for this idea. Instead, haploids were more susceptible to particular single-nucleotide mutations in late-replicating genomic regions, resulting in a ploidy difference in the spectrum of substitutions. In diploids we detect mutation rate variation among chromosomes in association with centromere location, a finding that is supported by published polymorphism data. Diploids are . CC-BY-NC-ND 4.0 International license peer-reviewed) is the author/funder. It is made available under a The copyright holder for this preprint (which was not . http://dx.doi.org/10.1101/250365 doi: bioRxiv preprint first posted online Jan. 19, 2018; 2 not simply doubled haploids; instead, our results predict that the spectrum of spontaneous mutations will substantially shape the dynamics of genome evolution in haploid and diploid populations.

show abstract

Section: Resultssupporting

confidence: 92%

The genome-wide rate and spectrum of spontaneous mutations differs between haploid and diploid yeast

Sharp

Sandell

James

et al. 2018

Preprint

View full text Add to dashboard Cite

show abstract

“…While the rate of aneuploidy in RDH54 + diploids (0.90 × 10 −4 ) is very similar to previous observations from diploid yeast (1.04 × 10 −4 ) (5), we found that rdh54Δ elevated the rate of aneuploidy fourfold overall and 4.5-fold within diploids ( Fig. 2; binomial test: P < 10 −4 ), indicating that RDH54 plays a role in mitotic chromosome segregation in addition to its role in meiosis (8,13), as suggested previously (14). Given the absence of aneuploidy in haploids, we cannot determine whether the increase in aneuploidy in rdh54Δ lines also applies to haploids or is unique to diploids.…”

Section: Resultssupporting

confidence: 91%

The genome-wide rate and spectrum of spontaneous mutations differ between haploid and diploid yeast

Sharp

Sandell

James

et al. 2018

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

140

199

View full text Add to dashboard Cite

By altering the dynamics of DNA replication and repair, alternative ploidy states may experience different rates and types of new mutations, leading to divergent evolutionary outcomes. We report a direct comparison of the genome-wide spectrum of spontaneous mutations arising in haploids and diploids following a mutation-accumulation experiment in the budding yeast Characterizing the number, types, locations, and effects of thousands of mutations revealed that haploids were more prone to single-nucleotide mutations (SNMs) and mitochondrial mutations, while larger structural changes were more common in diploids. Mutations were more likely to be detrimental in diploids, even after accounting for the large impact of structural changes, contrary to the prediction that mutations would have weaker effects, due to masking, in diploids. Haploidy is expected to reduce the opportunity for conservative DNA repair involving homologous chromosomes, increasing the insertion-deletion rate, but we found little support for this idea. Instead, haploids were more susceptible to SNMs in late-replicating genomic regions, resulting in a ploidy difference in the spectrum of substitutions. In diploids, we detect mutation rate variation among chromosomes in association with centromere location, a finding that is supported by published polymorphism data. Diploids are not simply doubled haploids; instead, our results predict that the spectrum of spontaneous mutations will substantially shape the dynamics of genome evolution in haploid and diploid populations.

show abstract

“…Since the recombination frequency between 40 bp repeats in yeast vegetative cultures was calculated as 2.9 × 10 −6 , with a 100-fold variation range, probably due to the intrinsic property of the selected sequence ( 28 ), we decided to test a set of constructs differing among them for the length of the repeat (from 40 to 800 bp) and for the length of the homologous ends (from 40 to 100 bp, see Table S1 in Supplementary Material). When the short repeat (40 bp) and the standard length of homology of a BIT cassette [65 bp ( 11 , 19 )] were used, the efficiency of the translocation NUP-TOP was very poor (0.6%) while when the repeats were extended up to 800 bp, even single site integration (SSI) events were favored against BIT [data not shown; for a detailed description of the differences between BIT and SSI pathways see Ref. ( 46 )].…”

Section: Resultsmentioning

confidence: 99%

“…The constructs were all amplified by High-Fidelity PCR (Kapa Biosystems), purified and verified by sequencing (BMR sequencing service, Padova). The total amount of cells per transformation was 2.2 × 10 8 and the efficiency (E) of each transformation was determined dividing the frequency (ν) for the DNA amount in microgram used in the transformation process (ν/μg DNA) ( 19 ).…”

Section: Methodsmentioning

confidence: 99%

“…BIT allows to precisely link together through a linear DNA cassette two targeted loci on different chromosomes, exploiting the Rad52/Rad54-dependent endogenous homologous recombination system (HRS) of the yeast cell ( 11 , 18 ). The efficiency of the resulting translocation event depends on the length of the homologous ends, on the selected yeast strain and on the DNA stability of the targeted loci ( 19 ). Using BIT followed by a pioneering pop-out technology we succeeded to generate an in vivo perfect fusion between the genes NUP145 (ortholog to human NUP98 ) and TOP2 (ortholog to human TOP2B ).…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Bridge-Induced Translocation between NUP145 and TOP2 Yeast Genes Models the Genetic Fusion between the Human Orthologs Associated With Acute Myeloid Leukemia

et al. 2017

Self Cite

View full text Add to dashboard Cite

In mammalian organisms liquid tumors such as acute myeloid leukemia (AML) are related to spontaneous chromosomal translocations ensuing in gene fusions. We previously developed a system named bridge-induced translocation (BIT) that allows linking together two different chromosomes exploiting the strong endogenous homologous recombination system of the yeast Saccharomyces cerevisiae. The BIT system generates a heterogeneous population of cells with different aneuploidies and severe aberrant phenotypes reminiscent of a cancerogenic transformation. In this work, thanks to a complex pop-out methodology of the marker used for the selection of translocants, we succeeded by BIT technology to precisely reproduce in yeast the peculiar chromosome translocation that has been associated with AML, characterized by the fusion between the human genes NUP98 and TOP2B. To shed light on the origin of the DNA fragility within NUP98, an extensive analysis of the curvature, bending, thermostability, and B-Z transition aptitude of the breakpoint region of NUP98 and of its yeast ortholog NUP145 has been performed. On this basis, a DNA cassette carrying homologous tails to the two genes was amplified by PCR and allowed the targeted fusion between NUP145 and TOP2, leading to reproduce the chimeric transcript in a diploid strain of S. cerevisiae. The resulting translocated yeast obtained through BIT appears characterized by abnormal spherical bodies of nearly 500 nm of diameter, absence of external membrane and defined cytoplasmic localization. Since Nup98 is a well-known regulator of the post-transcriptional modification of P53 target genes, and P53 mutations are occasionally reported in AML, this translocant yeast strain can be used as a model to test the constitutive expression of human P53. Although the abnormal phenotype of the translocant yeast was never rescued by its expression, an exogenous P53 was recognized to confer increased vitality to the translocants, in spite of its usual and well-documented toxicity to wild-type yeast strains. These results obtained in yeast could provide new grounds for the interpretation of past observations made in leukemic patients indicating a possible involvement of P53 in cell transformation toward AML.

show abstract

Bridge-Induced Chromosome Translocation in Yeast Relies upon a Rad54/Rdh54-Dependent, Pol32-Independent Pathway

Cited by 9 publications

References 64 publications

The genome-wide rate and spectrum of spontaneous mutations differs between haploid and diploid yeast

The genome-wide rate and spectrum of spontaneous mutations differs between haploid and diploid yeast

The genome-wide rate and spectrum of spontaneous mutations differ between haploid and diploid yeast

Bridge-Induced Translocation between NUP145 and TOP2 Yeast Genes Models the Genetic Fusion between the Human Orthologs Associated With Acute Myeloid Leukemia

Contact Info

Product

Resources

About