Biology subjects

Argueso, J. L.

Publications and source records attributed to Argueso, J. L..

13 recordsLinked to original sources

bioRxiv2026

Mechanical tuning of replication stress tolerance and genomic stability through the checkpoint mediator Mrc1

Mrc1 is a replication fork component that mediates communication between the replication checkpoint and fork progression. Here, we find that Mrc1 forms a dynamic mechanical linkage between CMG helicase and DNA Polymerase {varepsilon} that balances fork stability and flexibility. In its coupled state, Mrc1 bridges CMG helicase and the Pol2 C-terminal domain of Polymerase {varepsilon} to maintain helicase-polymerase coordination, promoting tolerance to replication stress but constraining fork remodeling. Upon checkpoint activation, Mec1/Rad53-dependent phosphorylation of Mrc1 shifts this equilibrium toward a loosened state, reducing coupling and enabling fork flexibility and lower mutation rate at the cost of reduced stress resistance.

biochemistry↗

bioRxiv2025

Candida glabrata replicating within macrophages experiences amino acid deprivation, DNA damage, and chromosome instability

Macrophages, the central players of innate immunity, control invading microbes by encapsulating them inside the phagosome, a nutrient-poor, reactive oxidant species-rich organelle. Nevertheless, some microbes, including the opportunistic yeast pathogen Candida glabrata, noted for its karyotype diversity, rapid evolution of antifungal drug resistance, and lack of meiosis, can survive and even replicate inside macrophages. However, it is not fully understood how C. glabrata responds to macrophage engulfment, and it is unknown how this presumably DNA-damaging environment influences the pathogens genome stability. In this study, we used comparative transcriptomics to identify amino acid starvation and DNA damage as conditions eliciting C. glabrata responses most similar to macrophage engulfment. Consistent with this, we found that C. glabrata intra-macrophage survival and replication require master regulator of amino acid biosynthesis GCN4 and functional DNA double-strand break repair. Furthermore, comet assays provided the first direct evidence for increased DNA breaks in intra-macrophage yeast, and pulse-field gel electrophoresis showed that chromosomal alterations occur frequently in macrophage-passaged C. glabrata. Interestingly, these alterations could not be resolved by long read DNA sequencing, suggesting that they involved highly complex repetitive regions. Finally, we identified several point mutations emerging during macrophage passaging and showed that among them, a frameshift in RME1 (repressor of meiosis in Saccharomyces cerevisiae), increased C. glabrata intra-macrophage fitness. Together, these analyses point to amino acid deprivation, reveal elevated DNA breakage and chromosome instability, and raise intriguing questions about the role of meiotic gene orthologs in C. glabrata persisting and replicating within macrophages.

microbiology↗

bioRxiv2025

Formation of chromosomal rearrangements in Saccharomyces cerevisiae diploids through regionally-biased non-allelic homologous recombination

In earlier studies, we optimized an assay system for the genome-wide detection of copy number variation (CNV) in diploid Saccharomyces cerevisiae cells, based on selection for formaldehyde plus copper (FA+Cu) resistance conferred by the amplification of a dosage-dependent reporter cassette, SFA1-CUP1. Our analyses identified a robust bias for terminal deletions of the right arm of Chr7 (Chr7R) associated with unbalanced translocations. This bias was observed at approximately constant strength across all three sites where the amplification reporter cassette was inserted, in CNV-carrying yeast clones derived both spontaneously and from mutagen-induced recombinogenic conditions. We conducted allelic mitotic recombination experiments to investigate the possibility of the presence of a fragile site on Chr7R, but the results disfavored this model, and instead indicated that the Chr7R bias applies only to non-allelic rearrangements. We validated the existence of a CNV formation bias at Chr7R through an orthologous NAHR competition approach that was independent of selection for FA+Cu resistance. Finally, we showed the in contrast to its high participation in NAHR as a recipient sequence, Chr7R becomes amplified as a translocation donor less frequently than other comparable regions of the genome. To begin unraveling the cause of this unusual behavior, we evaluated the effect of a set of candidate genes involved in chromatin mobility and sister chromatid cohesion on the rearrangement spectra involving Chr7R. We found that deletion mutations in some of these genes, particularly SAP30, attenuated the biased NAHR behavior. Taken together, our results suggested that although Chr7R is not inherently more prone to DNA breakage than other regions, once a DNA lesion is formed there, it has a higher propensity to undergo inappropriate repair leading to a chromosomal rearrangement.

genetics↗

bioRxiv2023

UV damage induces production of mitochondrial DNA fragments with specific length profiles

UV light is a potent mutagen that induces bulky DNA damage in the form of cyclobutane pyrimidine dimers (CPDs). In eukaryotic cells, photodamage and other bulky lesions occurring in nuclear genomes (nucDNAs) can be repaired through nucleotide excision repair (NER), where dual incisions on both sides of a damaged site precede the removal of a single-stranded oligonucleotide containing the damage. Mitochondrial genomes (mtDNAs) are also susceptible to damage from UV light, but current views hold that the only way to eliminate bulky DNA damage in mtDNAs is through mtDNA degradation. Damage-containing oligonucleotides excised during NER can be captured with anti-damage antibodies and sequenced (XR-seq) to produce high resolution maps of active repair locations following UV exposure. We analyzed previously published datasets from Arabidopsis thaliana, Saccharomyces cerevisiae, and Drosophila melanogaster to identify reads originating from the mtDNA (and plastid genome in A. thaliana). In A. thaliana and S. cerevisiae, the mtDNA-mapping reads have unique length distributions compared to the nuclear-mapping reads. The dominant fragment size was 26 nt in S. cerevisiae and 28 nt in A. thaliana with distinct secondary peaks occurring in 2-nt (S. cerevisiae) or 4-nt (A. thaliana) intervals. These reads also show a nonrandom distribution of di-pyrimidines (the substrate for CPD formation) with TT enrichment at positions 7-8 of the reads. Therefore, UV damage to mtDNA appears to result in production of DNA fragments of characteristic lengths and positions relative to the damaged location. We hypothesize that these fragments may reflect the outcome of a previously uncharacterized mechanism of NER-like repair in mitochondria or a programmed mtDNA degradation pathway.

genetics↗

bioRxiv2023

Delineation of two multi-invasion-induced rearrangement pathways that differently affect genome stability

Punctuated bursts of structural genomic variations (SVs) have been described in various organisms, but their etiology remains incompletely understood. Homologous recombination (HR) is a template-guided mechanism of repair of DNA double-strand breaks and stalled or collapsed replication forks. We recently identified a DNA break amplification and genome rearrangement pathway originating from the endonucleolytic processing of a multi-invasion (MI) DNA joint molecule formed during HR. Genome-wide sequencing approaches confirmed that multi-invasion-induced rearrangement (MIR) frequently leads to several repeat-mediated SVs and aneuploidies. Using molecular and genetic analysis, and a novel, highly sensitive proximity ligation-based assay for chromosomal rearrangement quantification, we further delineate two MIR sub-pathways. MIR1 is a universal pathway occurring in any sequence context, which generates secondary breaks and frequently leads to additional SVs. MIR2 occurs only if recombining donors exhibit substantial homology, and results in sequence insertion without additional break or SV. The most detrimental MIR1 pathway occurs late on a subset of persisting DNA joint molecules in a PCNA/Pol{delta}-independent manner, unlike recombinational DNA synthesis. This work provides a refined mechanistic understanding of these HR-based SV formation pathways and shows that complex repeat-mediated SVs can occur without displacement DNA synthesis. Sequence signatures for inferring MIR1 from long-read data are proposed.

genetics↗

bioRxiv2022

Bursts of genomic instability potentiate phenotypic and genomic diversification in Saccharomyces cerevisiae

How microbial cells leverage their phenotypic potential to survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. Stochastic phenotype switching, a particularly fascinating adaptive approach observed in numerous species across the tree of life, introduces phenotypic diversity into a population through mechanisms which have remained difficult to define. Here we describe our investigations into the mechanistic basis of colony morphology phenotype switching which occurs in populations of a pathogenic isolate of Saccharomyces cerevisiae, YJM311. We observed that clonal populations of YJM311 cells produce variant colonies that display altered morphologies and, using whole genome sequence analysis, discovered that these variant clones harbored an exceptional collection of karyotypes newly altered by de novo structural genomic variations (SVs). Overall, our analyses indicate that copy number alterations, more often than changes in allelic identity, provide the causative basis of this phenotypic variation. Individual variants carried between 1 and 16 de novo copy number variations, most of which were whole chromosomal aneuploidies. Notably, the inherent stability of the diploid YJM311 genome is comparable to that of domesticated laboratory strains, indicating that the collections of SVs harbored by variant clones did not arise by a chronic chromosomal instability (CIN) mechanism. Rather, variant clones acquired these complex karyotypic configurations simultaneously, during stochastic and transient episodes of punctuated systemic genomic instability (PSGI). Surprisingly, we found that the majority of these highly altered variant karyotypes were propagated with perfect fidelity in long-term passaging experiments, demonstrating that high aneuploidy burdens can often be conducive with prolonged genomic instability. Together, our results demonstrate that colony morphology switching in YJM311 is driven by a stochastic process in which genome stability and plasticity are integrally coupled to phenotypic heterogeneity. Consequently, this system simultaneously introduces both phenotypic and genomic variation into a population of cells, which can, in turn perpetuate population diversity for many generations thereafter.

genomics↗

bioRxiv2021

Genomic characterization of a pathogenic isolate of Saccharomyces cerevisiae reveals an extensive and dynamic landscape of structural variation

The budding yeast Saccharomyces cerevisiae has been extensively characterized for many decades and is a critical resource for the study of numerous facets of eukaryotic biology. Recently, the analysis of whole genome sequencing data from over 1000 natural isolates of S. cerevisiae has provided critical insights into the evolutionary landscape of this species by revealing a population structure comprised of numerous genomically diverse lineages. These survey-level analyses have been largely devoid of structural genomic information, mainly because short read sequencing is not suitable for detailed characterization of genomic architecture. Consequently, we still lack a complete perspective of the genomic variation the exists within the species. Single molecule long read sequencing technologies, such as Oxford Nanopore and PacBio, provide sequencing-based approaches with which to rigorously define the structure of a genome, and have empowered yeast geneticists to explore this poorly described realm of eukaryotic genomics. Here, we present the comprehensive genomic structural analysis of a pathogenic isolate of S. cerevisiae, YJM311. We used long read sequence analysis to construct a haplotype-phased, telomere-to-telomere length assembly of the YJM311 diploid genome and characterized the structural variations (SVs) therein. We discovered that the genome of YJM311 contains significant intragenomic structural variation, some of which imparts notable consequences to the genomic stability and developmental biology of the strain. Collectively, we outline a new methodology for creating accurate haplotype-phased genome assemblies and highlight how such genomic analyses can define the structural architectures of S. cerevisiae isolates. It is our hope that through continued structural characterization of S. cerevisiae genomes, such as we have reported here for YJM311, we will comprehensively advance our understanding of eukaryotic genome structure-function relationships, structural diversity, and evolution.

genomics↗

bioRxiv2021

Systemic aneuploidization events drive phenotype switching in Saccharomyces cerevisiae

How cells leverage their phenotypic potential to adapt and survive in a changing environment is a complex biological problem, with important implications for pathogenesis and species evolution. One particularly fascinating adaptive approach is the bet hedging strategy known as phenotype switching, which introduces phenotypic variation into a population through stochastic processes. Phenotype switching has long been observed in species across the tree of life, yet the mechanistic causes of switching in these organisms have remained difficult to define. Here we describe the causative basis of colony morphology phenotype switching which occurs among cells of the pathogenic isolate of Saccharomyces cerevisiae, YJM311. From clonal populations of YJM311 cells grown in identical conditions, we identified colonies which displayed altered colony architectures, yet could revert to the wild-type morphology after passaging. Whole genome sequence analysis revealed that these variant clones had all acquired whole chromosome copy number alterations (i.e., aneuploidies). Cumulatively, the variant clones we characterized harbored an exceptional spectrum of karyotypic alterations, with individual variants carrying between 1 and 16 aneuploidies. Most variants harbored unique collections of aneuploidies, indicating that numerous distinct karyotypes could manifest in the same morphological variation. Intriguingly, the genomic stability of these newly aneuploid variant clones modulated how often cells reverted back to the wild-type phenotypic state. We found that such revertant switches were also driven by chromosome missegregation events, and in some cases occurred through a return to euploidy. Together, our results demonstrate that colony morphology switching in this yeast strain is driven by stochastic and systemic aneuploidization events. These findings add an important new perspective to our current understanding of phenotype switching and bet hedging strategies, as well as how environmental pressures perpetuate organismal adaption and genome evolution.

genomics↗

bioRxiv2020

A new experimental system to study meiotic recurrent non-allelic homologous recombination in budding yeast

In humans, de novo recurrent copy number variations (CNVs) often arise during meiosis from non-allelic homologous recombination (NAHR) between low copy repeat elements (LCRs). These chromosomal rearrangements are responsible for a wide variety of genomic disorders involving duplication or deletion of dose-sensitive genes. The precise factors that steer meiotic cells toward this detrimental recombination pathway are not fully understood. To create a model for the investigation of LCR-mediated CNV mechanisms, we developed a diploid experimental system in Saccharomyces cerevisiae. We modified the right arm of chromosome V through the introduction of engineered LCRs: duplicated 5 to 35 kb segments of yeast DNA flanking single copy interstitial spacers, analogously to the meiotic NAHR substrates that exist in humans. Phenotypic markers, including a copy number reporter, were inserted within the interstitial spacer. Their segregation in the haploid meiotic progeny was used to phenotypically identity and classify recurrent CNV events. This system allowed us to measure the effects of LCR size on the frequency of meiotic de novo recurrent CNV formation, and to determine the relative proportions of each of the three main NAHR classes: interhomolog, intersister, and intrachromatid. The frequency of CNV increased as the LCRs became larger, and interhomolog NAHR was overrepresented relative to the two other classes. We showed that this experimental system directly mimics the features of de novo recurrent CNVs reported in human disease, thus it represents a promising tool for the discovery and characterization of conserved cellular factors and environmental exposures that can modulate meiotic NAHR.

genetics↗

bioRxiv2020

Characterization of systemic genomic instability in budding yeast

Conventional models of genome evolution are centered around the principle that mutations form independently of each other and build up slowly over time. We characterized the occurrence of bursts of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, providing support for an additional non-independent and faster mode of mutation accumulation. We initially characterized a yeast clone isolated for carrying an LOH event at a specific chromosome site, and surprisingly, found that it also carried multiple unselected rearrangements elsewhere in its genome. Whole genome analysis of over 100 additional clones selected for carrying primary LOH tracts revealed that they too contained unselected structural alterations more often than control clones obtained without any selection. We also measured the rates of coincident LOH at two different chromosomes and found that double LOH formed at rates 14-150 fold higher than expected if the two underlying single LOH events occurred independently of each other. These results were consistent across different strain backgrounds, and in mutants incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population can experience discrete episodes of systemic genomic instability, when the entire genome becomes vulnerable and multiple chromosomal alterations can form over a narrow time window. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in the cancer and genomic disorder contexts. The experimental model we describe provides a system to further dissect the fundamental biological processes responsible for punctuated bursts of structural genomic variation. SIGNIFICANCE STATEMENTMutations are generally thought to accumulate independently and gradually over many generations. Here, we combined complementary experimental approaches in budding yeast to track the appearance of chromosomal changes resulting in loss-of-heterozygosity (LOH). In contrast to the prevailing model, our results provide evidence for the existence of a path for non-independent accumulation of multiple chromosomal alteration events over few generations. These results are analogous to recent reports of bursts of genomic instability in human cells. The experimental model we describe provides a system to further dissect the fundamental biological processes underlying such punctuated bursts of mutation accumulation.

genetics↗

bioRxiv2020

Punctuated Aneuploidization of the Budding Yeast Genome

Remarkably complex patterns of aneuploidy have been observed in the genomes of many eukaryotic cell types, ranging from brewing yeasts to tumor cells (1, 2). Such aberrant karyotypes are generally thought to take shape progressively over many generations, but evidence also suggests that genomes may undergo faster modes of evolution (2, 3). Here, we used diploid Saccharomyces cerevisiae cells to investigate the dynamics with which aneuploidies arise. We found that cells selected for the loss of a single chromosome often acquired additional unselected aneuploidies concomitantly. The degrees to which these genomes were altered fell along a spectrum, ranging from simple events affecting just a single chromosome, to systemic events involving many. The striking complexity of karyotypes arising from systemic events, combined with the high frequency at which we detected them, demonstrates that cells can rapidly achieve highly altered genomic configurations during temporally restricted episodes of genomic instability.

genomics↗

bioRxiv2018

The Chromatin-binding Protein Spn1 contributes to Genome Instability in Saccharomyces cerevisiae

Cells expend a large amount of energy to maintain their DNA sequence. DNA repair pathways, cell cycle checkpoint activation, proofreading polymerases, and chromatin structure are ways in which the cell minimizes changes to the genome. During replication, the DNA damage tolerance pathway allows the replication forks to bypass damage on the template strand. This avoids prolonged replication fork stalling, which can contribute to genome instability. The DNA damage tolerance pathway includes two sub-pathways: translesion synthesis and template switch. Post-translational modification of PCNA and the histone tails, cell cycle phase, and local DNA structure have all been shown to influence sub-pathway choice. Chromatin architecture contributes to maintaining genome stability by providing physical protection of the DNA and by regulating DNA processing pathways. As such, chromatin-binding factors have been implicated in maintaining genome stability. Using Saccharomyces cerevisiae, we examined the role of Spn1, a chromatin binding and transcription elongation factor, in DNA damage tolerance. Expression of a mutant allele of SPN1 results in increased resistance to the DNA damaging agent methyl methanesulfonate, lower spontaneous and damage-induced mutation rates, along with increased chronological lifespan. We attribute these effects to an increased usage of the template switch branch of the DNA damage tolerance pathway in the spn1 strain. This provides evidence for a role of wild type Spn1 in promoting genome instability, as well as having ties to overcoming replication stress and contributing to chronological aging.

genetics↗

bioRxiv2017

Mitotic systemic genomic instability in yeast

Conventional models of genome evolution generally include the assumption that mutations accumulate gradually and independently over time. We characterized the occurrence of sudden spikes in the accumulation of genome-wide loss-of-heterozygosity (LOH) in Saccharomyces cerevisiae, suggesting the existence of a mitotic systemic genomic instability process (mitSGI). We characterized the emergence of a rough colony morphology phenotype resulting from an LOH event spanning a specific locus (ACE2/ace2-A7). Surprisingly, half of the clones analyzed also carried unselected secondary LOH tracts elsewhere in their genomes. The number of secondary LOH tracts detected was 20-fold higher than expected assuming independence between mutational events. Secondary LOH tracts were not detected in control clones without a primary selected LOH event. We then measured the rates of single and double LOH at different chromosome pairs and found that coincident LOH accumulated at rates 30-100 fold higher than expected if the two underlying single LOH events occurred independently. These results were consistent between two different strain backgrounds, and in mutant strains incapable of entering meiosis. Our results indicate that a subset of mitotic cells within a population experience systemic genomic instability episodes, resulting in multiple chromosomal rearrangements over one or few generations. They are reminiscent of early reports from the classic yeast genetics literature, as well as recent studies in humans, both in the cancer and genomic disorder contexts, all of which challenge the idea of gradual accumulation of structural genomic variation. Our experimental approach provides a model to further dissect the fundamental mechanisms responsible for mitSGI.\n\nSIGNIFICANCE STATEMENTPoint mutations and alterations in chromosome structure are generally thought to accumulate gradually and independently over many generations. Here, we combined complementary genetic approaches in budding yeast to track the appearance of chromosomal changes resulting in loss-of-heterozygosity (LOH). Contrary to expectations, our results provided evidence for the occurrence of non-independent accumulation of multiple LOH events over one or a few cell generations. These results are analogous to recent reports of bursts of chromosomal instability in humans. Our experimental approach provides a framework to further dissect the fundamental mechanisms underlying systemic chromosomal instability processes, including in the human cancer and genomic disorder contexts.

genetics↗