scholarly journals Rate volatility and asymmetric segregation diversify mutation burden in mutator cells

2020 ◽  
Author(s):  
I.T. Dowsett ◽  
J. Sneeden ◽  
B.J. Olson ◽  
J. McKay-Fleisch ◽  
E. McAuley ◽  
...  
Keyword(s):  
Mismatch Repair ◽  
Dna Polymerase ◽  
Count Data ◽  
Yeast Cells ◽  
Cancer Evolution ◽  
Mendelian Segregation ◽  
Replication Errors ◽  
Daughter Cells ◽  
Mutation Burden ◽  
Mother And Daughter

Mutations that compromise mismatch repair (MMR) or DNA polymerase exonuclease domains produce mutator phenotypes capable of fueling cancer evolution. Tandem defects in these pathways dramatically increase mutation rate. Here, we model how mutator phenotypes expand genetic heterogeneity in budding yeast cells using a single-cell resolution approach that tallies all replication errors arising from individual divisions. The distribution of count data from cells lacking MMR and polymerase proofreading was broader than expected for a single rate, consistent with volatility of the mutator phenotype. The number of mismatches that segregated to the mother and daughter cells after the initial round of replication co-varied, suggesting that mutagenesis in each division is governed by a different underlying rate. The distribution of “fixed” mutation counts that cells inherit is further broadened by an unequal sharing of mutations due to semiconservative replication and Mendelian segregation. Modeling suggests that this asymmetric segregation may diversify mutation burden in mutator-driven tumors.

scholarly journals Rate volatility and asymmetric segregation diversify mutation burden in cells with mutator alleles

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Ian T. Dowsett ◽  
Jessica L. Sneeden ◽  
Branden J. Olson ◽  
Jill McKay-Fleisch ◽  
Emma McAuley ◽  
...  
Keyword(s):  
Mutation Rate ◽  
S Phase ◽  
Cancer Evolution ◽  
Replication Errors ◽  
Daughter Cells ◽  
Genome Wide ◽  
Mutation Burden ◽  
Mother And Daughter ◽  
Mother Cells ◽  
In Cells

AbstractMutations that compromise mismatch repair (MMR) or DNA polymerase ε or δ exonuclease domains produce mutator phenotypes capable of fueling cancer evolution. Here, we investigate how combined defects in these pathways expands genetic heterogeneity in cells of the budding yeast, Saccharomyces cerevisiae, using a single-cell resolution approach that tallies all mutations arising from individual divisions. The distribution of replication errors present in mother cells after the initial S-phase was broader than expected for a single uniform mutation rate across all cell divisions, consistent with volatility of the mutator phenotype. The number of mismatches that then segregated to the mother and daughter cells co-varied, suggesting that each division is governed by a different underlying genome-wide mutation rate. The distribution of mutations that individual cells inherit after the second S-phase is further broadened by the sequential actions of semiconservative replication and mitotic segregation of chromosomes. Modeling suggests that this asymmetric segregation may diversify mutation burden in mutator-driven tumors.

scholarly journals Complex Minisatellite Rearrangements Generated in the Total or Partial Absence of Rad27/hFEN1 Activity Occur in a Single Generation and Are Rad51 and Rad52 Dependent

2006 ◽  
Vol 26 (17) ◽  
pp. 6675-6689 ◽  
Author(s):  
Judith Lopes ◽  
Cyril Ribeyre ◽  
Alain Nicolas
Keyword(s):  
Pedigree Analysis ◽  
Model System ◽  
Yeast Cells ◽  
Single Generation ◽  
Daughter Cells ◽  
Double Deletion ◽  
Mother And Daughter ◽  
Complex Events ◽  
High Degree ◽  
Insight Into

ABSTRACT Genomes contain tandem repeat blocks that are at risk of expansion or contraction. The mechanisms of destabilization of the human minisatellite CEB1 (arrays of 36- to 43-bp repeats) were investigated in a previously developed model system, in which CEB1-0.6 (14 repeats) and CEB1-1.8 (42 repeats) alleles were inserted into the genome of Saccharomyces cerevisiae. As in human cells, CEB1 is stable in mitotically growing yeast cells but is frequently rearranged in the absence of the Rad27/hFEN1 protein involved in Okazaki fragments maturation. To gain insight into this mode of destabilization, the CEB1-1.8 and CEB1-0.6 human alleles and 47 rearrangements derived from a CEB1-1.8 progenitor in rad27Δ cells were sequenced. A high degree of polymorphism of CEB1 internal repeats was observed, attesting to a large variety of homology-driven rearrangements. Simple deletion, double deletion, and highly complex events were observed. Pedigree analysis showed that all rearrangements, even the most complex, occurred in a single generation and were inherited equally by mother and daughter cells. Finally, the rearrangement frequency was found to increase with array size, and partial complementation of the rad27Δ mutation by hFEN1 demonstrated that the production of novel CEB1 alleles is Rad52 and Rad51 dependent. Instability can be explained by an accumulation of unresolved flap structures during replication, leading to the formation of recombinogenic lesions and faulty repair, best understood by homology-dependent synthesis-strand displacement and annealing.

scholarly journals Hsp104 Overexpression Cures Saccharomyces cerevisiae [ PSI + ] by Causing Dissolution of the Prion Seeds

2014 ◽  
Vol 13 (5) ◽  
pp. 635-647 ◽  
Author(s):  
Yang-Nim Park ◽  
Xiaohong Zhao ◽  
Yang-In Yim ◽  
Horia Todor ◽  
Robyn Ellerbrock ◽  
...  
Keyword(s):  
Molecular Chaperone ◽  
Fluorescent Protein ◽  
Yeast Cells ◽  
Yeast Prion ◽  
Yeast Prions ◽  
Daughter Cells ◽  
Amyloid Aggregates ◽  
Mother And Daughter ◽  
Green Fluorescent ◽  
Kinetics Of

ABSTRACT The [ PSI + ] yeast prion is formed when Sup35 misfolds into amyloid aggregates. [ PSI + ], like other yeast prions, is dependent on the molecular chaperone Hsp104, which severs the prion seeds so that they pass on as the yeast cells divide. Surprisingly, however, overexpression of Hsp104 also cures [ PSI + ]. Several models have been proposed to explain this effect: inhibition of severing, asymmetric segregation of the seeds between mother and daughter cells, and dissolution of the prion seeds. First, we found that neither the kinetics of curing nor the heterogeneity in the distribution of the green fluorescent protein (GFP)-labeled Sup35 foci in partially cured yeast cells is compatible with Hsp104 overexpression curing [ PSI + ] by inhibiting severing. Second, we ruled out the asymmetric segregation model by showing that the extent of curing was essentially the same in mother and daughter cells and that the fluorescent foci did not distribute asymmetrically, but rather, there was marked loss of foci in both mother and daughter cells. These results suggest that Hsp104 overexpression cures [ PSI + ] by dissolution of the prion seeds in a two-step process. First, trimming of the prion seeds by Hsp104 reduces their size, and second, their amyloid core is eliminated, most likely by proteolysis.

scholarly journals The fitness cost of mismatch repair mutators in Saccharomyces cerevisiae: partitioning the mutational load

2019 ◽  
Author(s):  
Benjamin Galeota-Sprung ◽  
Breanna Guindon ◽  
Paul Sniegowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mismatch Repair ◽  
Separate Experiment ◽  
Mutational Load ◽  
Deleterious Mutations ◽  
Competitive Fitness ◽  
Daughter Cells ◽  
Point Estimates ◽  
Mother And Daughter ◽  
Mean Fitness

AbstractMutational load is the depression in a population’s mean fitness that results from the continual influx of deleterious mutations. Here, we directly estimate the mutational load in a population of haploid Saccharomyces cerevisiae that are deficient for mismatch repair. We partition the load in haploids into two components. To estimate the load due to nonlethal mutations, we measure the competitive fitness of hundreds of randomly selected clones from both mismatch repair-deficient and - proficient populations. Computation of the mean clone fitness for the mismatch repair-deficient strain permits an estimation of the nonlethal load, and the histogram of fitness provides an interesting visualization of a loaded population. In a separate experiment, in order to estimate the load due to lethal mutations (i.e. the lethal mutation rate), we manipulate thousands of individual pairs of mother and daughter cells and track their fates. These two approaches yield point estimates for the two contributors to load, and the addition of these estimates is nearly equal to the separately measured short-term competitive fitness deficit for the mismatch repair-deficient strain. This correspondence suggests that there is no need to invoke direct fitness effects to explain the fitness difference between mismatch repair-deficient and - proficient strains. Assays in diploids are consistent with deleterious mutations in diploids tending towards recessivity. These results enhance our understanding of mutational load, a central population genetics concept, and we discuss their implications for the evolution of mutation rates.

scholarly journals Solid-phase inclusion as a mechanism for regulating unfolded proteins in the mitochondrial matrix

2020 ◽  
Vol 6 (32) ◽  
pp. eabc7288
Author(s):  
Linhao Ruan ◽  
Joshua T. McNamara ◽  
Xi Zhang ◽  
Alexander Chih-Chieh Chang ◽  
Jin Zhu ◽  
...  
Keyword(s):  
Solid Phase ◽  
Tricarboxylic Acid Cycle ◽  
Yeast Cells ◽  
Mitochondrial Proteins ◽  
Mitochondrial Matrix ◽  
Unfolded Proteins ◽  
Daughter Cells ◽  
Contact Sites ◽  
Age Dependent ◽  
Mother And Daughter

Proteostasis declines with age, characterized by the accumulation of unfolded or damaged proteins. Recent studies suggest that proteins constituting pathological inclusions in neurodegenerative diseases also enter and accumulate in mitochondria. How unfolded proteins are managed within mitochondria remains unclear. Here, we found that excessive unfolded proteins in the mitochondrial matrix of yeast cells are consolidated into solid-phase inclusions, which we term deposits of unfolded mitochondrial proteins (DUMP). Formation of DUMP occurs in mitochondria near endoplasmic reticulum–mitochondria contact sites and is regulated by mitochondrial proteins controlling the production of cytidine 5′-diphosphate–diacylglycerol. DUMP formation is age dependent but accelerated by exogenous unfolded proteins. Many enzymes of the tricarboxylic acid cycle were enriched in DUMP. During yeast cell division, DUMP formation is necessary for asymmetric inheritance of damaged mitochondrial proteins between mother and daughter cells. We provide evidence that DUMP-like structures may be induced by excessive unfolded proteins in human cells.

scholarly journals A novel factor essential for unconventional secretion of chitinase Cts1

2020 ◽  
Author(s):  
Michèle Reindl ◽  
Janpeter Stock ◽  
Kai P. Hussnaetter ◽  
Aycin Genc ◽  
Andreas Brachmann ◽  
...  
Keyword(s):  
Cell Division ◽  
Underlying Mechanism ◽  
Yeast Cells ◽  
Subcellular Targeting ◽  
Uv Mutagenesis ◽  
Daughter Cells ◽  
Secretion Pathway ◽  
Unconventional Secretion ◽  
Mother And Daughter ◽  
Two Hybrid

AbstractSubcellular targeting of proteins is essential to orchestrate cytokinesis in eukaryotic cells. During cell division of Ustilago maydis, for example, chitinases must be specifically targeted to the fragmentation zone at the site of cell division to degrade remnant chitin and thus separate mother and daughter cells. Chitinase Cts1 is exported to this location via an unconventional secretion pathway putatively operating in a lock-type manner. The underlying mechanism is largely unexplored. Here, we applied a forward genetic screen based on UV mutagenesis to identify components essential for Cts1 export. The screen revealed a novel factor termed Jps1 lacking known protein domains. Deletion of the corresponding gene confirmed its essential role for Cts1 secretion. Localization studies demonstrated that Jps1 colocalizes with Cts1 in the fragmentation zone of dividing yeast cells. While loss of Jps1 leads to exclusion of Cts1 from the fragmentation zone and strongly reduced unconventional secretion, deletion of the chitinase does not disturb Jps1 localization. Yeast-two hybrid experiments suggest that the two proteins interact. In essence, we identified a novel component of unconventional secretion that functions in the fragmentation zone to enable export of Cts1. We hypothesize that Jps1 acts as an anchoring factor, supporting the proposed novel lock-type mechanism of unconventional secretion.

scholarly journals Spontaneous mutation in Escherichia coli containing the dnaE911 DNA polymerase antimutator allele.

Genetics ◽  
1994 ◽  
Vol 138 (2) ◽  
pp. 263-270 ◽  
Author(s):  
A R Oller ◽  
R M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Dna Polymerase ◽  
Spontaneous Mutation ◽  
Gene Encoding ◽  
Replication Errors ◽  
Lac Operator ◽  
Laci Gene ◽  
Dna Replication Errors

Abstract We have previously isolated mutants of Escherichia coli that replicate their DNA with increased fidelity. These mutants have a mutation in the dnaE gene, encoding the alpha subunit of DNA polymerase III. They were isolated in a mismatch-repair-defective mutL background, in which mutations can be considered to represent uncorrected DNA replication errors. In the present study we analyze the effect of one of these alleles, dnaE911, on spontaneous mutagenesis in a mismatch-repair-proficient background. In this background, spontaneous mutations may be the sum of uncorrected replication errors and mutations resulting from other pathways. Hence, the effect of the dnaE allele may provide insights into the contribution of uncorrected DNA replication errors to spontaneous mutation. The data show that dnaE911 decreases the level of Rifr, lacI and galK mutations in this background by 1.5-2-fold. DNA sequencing of 748 forward mutants in the lacI gene reveals that this effect has a clear specificity. Transversions are decreased by approximately 3-fold, whereas transitions, frameshifts, deletions and duplications remain essentially unchanged. Among the transversions, A.T-->T.A are affected most strongly (approximately 6-fold). In addition to this effect on transversions within the lacI gene, one previously recognized A.T-->G.C base-pair substitution hotspot in the lac operator is also reduced (approximately 5-fold). The data are discussed in the light of the role of DNA replication errors in spontaneous mutation, as well as other possible explanations for the observed antimutator effects.

scholarly journals A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae.

1993 ◽  
Vol 13 (3) ◽  
pp. 1489-1496 ◽  
Author(s):  
S W Houtteman ◽  
R T Elder
Keyword(s):  
Dna Polymerase ◽  
Mutant Gene ◽  
Recessive Mutation ◽  
Wild Type ◽  
Dna Polymerase Delta ◽  
Autonomously Replicating Sequence ◽  
Plasmid Segregation ◽  
Daughter Cells ◽  
Mother And Daughter ◽  
Mutant Cells

Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

Difference in generation times for mother and daughter cells in yeasts

1978 ◽  
Vol 24 (7) ◽  
pp. 827-833 ◽  
Author(s):  
T. W. Flegel
Keyword(s):  
Growth Characteristic ◽  
Time Lapse ◽  
Yeast Cells ◽  
Daughter Cells ◽  
Yeast Strains ◽  
Generation Times ◽  
Spot Check ◽  
Mother And Daughter ◽  
The Mean ◽  
Mother Cells

Fruitless attempts to synchronize haploid yeast cells from the Phragmobasidiomycete Sirobasidium magnum led to the discovery that the mother and daughter cells (MDC) had greatly different generation times. Time-lapse photographic sequences of budding showed that the mean generation time for daughter cells was more than three times greater than that for mother cells. This growth characteristic could be determined by a spot check of the microcolony pattern on agar. Using such a check, yeast strains of Rhodotorula (Rhodosporidium) and Cryptococcus that were tested demonstrated relative MDC equivalence while those of Sporobolomyces, Bullera, and Tremella showed MDC non-equivalence in varying degrees.

scholarly journals A DNA polymerase mutation that suppresses the segregation bias of an ARS plasmid in Saccharomyces cerevisiae

1993 ◽  
Vol 13 (3) ◽  
pp. 1489-1496
Author(s):  
S W Houtteman ◽  
R T Elder
Keyword(s):  
Dna Polymerase ◽  
Mutant Gene ◽  
Recessive Mutation ◽  
Wild Type ◽  
Dna Polymerase Delta ◽  
Autonomously Replicating Sequence ◽  
Plasmid Segregation ◽  
Daughter Cells ◽  
Mother And Daughter ◽  
Mutant Cells

Yeast autonomously replicating sequence (ARS) plasmids exhibit an unusual segregation pattern during mitosis. While the nucleus divides equally into mother and daughter cells, all copies of the ARS plasmid will often remain in the mother cell. A screen was designed to isolate mutations that suppress this segregation bias. A plasmid with a weak ARS (wARS) that displayed an extremely high segregation bias was constructed. When cells were grown under selection for the wARS plasmid, the resulting colonies grew slowly and had abnormal morphology. A spontaneous recessive mutation that restored normal colony morphology was identified. This mutation suppressed plasmid segregation bias, as indicated by the increased stability of the wARS plasmid in the mutant cells even though the plasmid was present at a lower copy number. An ARS1 plasmid was also more stable in mutant cells than in wild-type cells. The wild-type allele for this mutant gene was cloned and identified as POL delta (CDC2). This gene encodes DNA polymerase delta, which is essential for DNA replication. These results indicate that DNA polymerase delta plays some role in causing the segregation bias of ARS plasmids.

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