Lack of G1/S control destabilizes the yeast genome via replication stress-induced DSBs and illegitimate recombination

Król, Kamil; Antoniuk-Majchrzak, Justyna; Skoneczny, Marek; Sieńko, Marzena; Jendrysek, Justyna; Rumieńczyk, Izabela; Halas, Agnieszka; Kurlandzka, Anna; Skoneczna, Adrianna

doi:10.1242/jcs.226480

Cited by 9 publications

(14 citation statements)

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“…The DNA content of yeast cells was measured by flow cytometry as previously described [83], with modifications restricting cells aggregations. About 10 7 cells from initial and final yeast cultures were spun down (19,300 g for 1 min) and subjected to permeabilization and fixation via suspension in 1 ml of chilled (− 20 °C) 80% ethanol (Polmos, Warsaw, Poland).…”

Section: Dna Content Analysis By Flow Cytometrymentioning

confidence: 99%

Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast

et al. 2021

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Background The impact of genetic interaction networks on evolution is a fundamental issue. Previous studies have demonstrated that the topology of the network is determined by the properties of the cellular machinery. Functionally related genes frequently interact with one another, and they establish modules, e.g., modules of protein complexes and biochemical pathways. In this study, we experimentally tested the hypothesis that compensatory evolutionary modifications, such as mutations and transcriptional changes, occur frequently in genes from perturbed modules of interacting genes. Results Using Saccharomyces cerevisiae haploid deletion mutants as a model, we investigated two modules lacking COG7 or NUP133, which are evolutionarily conserved genes with many interactions. We performed laboratory evolution experiments with these strains in two genetic backgrounds (with or without additional deletion of MSH2), subjecting them to continuous culture in a non-limiting minimal medium. Next, the evolved yeast populations were characterized through whole-genome sequencing and transcriptome analyses. No obvious compensatory changes resulting from inactivation of genes already included in modules were identified. The supposedly compensatory inactivation of genes in the evolved strains was only rarely observed to be in accordance with the established fitness effect of the genetic interaction network. In fact, a substantial majority of the gene inactivations were predicted to be neutral in the experimental conditions used to determine the interaction network. Similarly, transcriptome changes during continuous culture mostly signified adaptation to growth conditions rather than compensation of the absence of the COG7, NUP133 or MSH2 genes. However, we noticed that for genes whose inactivation was deleterious an upregulation of transcription was more common than downregulation. Conclusions Our findings demonstrate that the genetic interactions and the modular structure of the network described by others have very limited effects on the evolutionary trajectory following gene deletion of module elements in our experimental conditions and has no significant impact on short-term compensatory evolution. However, we observed likely compensatory evolution in functionally related (albeit non-interacting) genes.

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Section: Dna Content Analysis By Flow Cytometrymentioning

confidence: 99%

Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast

et al. 2021

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“…A similar effect was documented for cells lacking the transcription factor Swi6, which controls the expression of G1/S transition genes, resulting in limitation of proteins involved in DNA replication and repair and a prolonged cell cycle. Their survival is enhanced by Rad51-dependent illegitimate recombination [93]. Similar phenotypes were also described for human cells, in which mutation in the KRAS proto-oncogene caused replication fork stalling, DNA lesion accumulation and increased abundance of various proteins, including Rad51, which is obligatory for the survival of these mutant cells [144].…”

Section: Discussionmentioning

confidence: 73%

“…This is a highly conserved mechanism for the repair of DNA double-strand breaks (DSBs) and recovery of stalled or collapsed replication forks [90–92]. However, although recombination is thought to be error free, this mechanism is also potentially mutagenic, e.g., recombination can promote the instability of repeated DNA sequences [27] or cause genome rearrangements in the cells under constant replication stress [93].…”

Section: Resultsmentioning

confidence: 99%

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Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism

et al. 2019

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Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction.

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“…But the abundance of Rad51 in the cell strongly depends on its half-life life, which should be tightly controlled to regulate and resume the homologous recombination repair pathway. Indeed, the high level of Rad51 during replication stress leads to an increased usage of illegitimate recombination causing frequent genome rearrangement (21).…”

Section: Introductionmentioning

confidence: 99%

Intracellular level of S. cerevisiae Rad51 is regulated via proteolysis in a SUMO- and ubiquitin-dependent manner

Antoniuk-Majchrzak

Długajczyk

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et al. 2022

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Among various DNA lesions, the DNA double-strand breaks are particularly deleterious; especially, when an error-free repair pathway is unavailable, and the cell takes the risk of using the error-prone recombination pathways to repair the DNA breaks, resume the cell cycle, and continue growth. The latter comes at the expense of decreased well-being of the cells due to genome rearrangements. One of the major players involved in recombinational repair of DNA damage is Rad51 recombinase, a protein responsible for presynaptic complex formation. We previously noticed that the level of this protein is strongly increased when illegitimate recombination is engaged in repair. The regulation of Rad51 protein turnover is not known; therefore, we decided to look closer at this issue because we expect that an excessively high level of Rad51 may lead to genome instability. Here we show that the level of Rad51 is regulated via the ubiquitin-dependent proteolytic pathway. The ubiquitination of Rad51 depends on multiple E3 enzymes, including SUMO-targeted ubiquitin ligases. We also demonstrate that Rad51 can be modified by both ubiquitin and SUMO. Moreover, these modifications may lead to opposite effects. Ubiquitin-dependent degradation depends on Rad6, Rad18, Slx8, Dia2 and the anaphase-promoting complex. Rsp5-dependent ubiquitination leads to Rad51 stabilization.

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Lack of G1/S control destabilizes the yeast genome via replication stress-induced DSBs and illegitimate recombination

Cited by 9 publications

References 74 publications

Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast

Genetic interaction network has a very limited impact on the evolutionary trajectories in continuous culture-grown populations of yeast

Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism

Intracellular level of S. cerevisiae Rad51 is regulated via proteolysis in a SUMO- and ubiquitin-dependent manner

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