Biology subjects

Ollodart, A. R.

Publications and source records attributed to Ollodart, A. R..

4 recordsLinked to original sources

bioRxiv2023

Multidimensional proteomics identifies molecular trajectories of cellular aging and rejuvenation

The declining capacity of cells to maintain a functional proteome is a major driver of cellular dysfunction and decreased fitness in aging. Here we assess the impact of aging on multiple proteome dimensions, which are reflective of function, across the replicative lifespan of Saccharomyces cerevisiae. We quantified protein abundance, protein turnover, protein thermal stability, and protein phosphorylation in mother yeast cells and their derived progeny at different ages. We find progressive and cumulative proteomic alterations that are reflective of dysregulation of complex assemblies, mitochondrial remodeling, post-translational activation of the AMPK/Snf1 energy sensor in mother cells, and an overall shift from biosynthetic to energy-metabolic processes. Our multidimensional proteomic study systematically corroborates previous findings of asymmetric segregation and daughter cell rejuvenation, and extends these concepts to protein complexes, protein phosphorylation, and activation of signaling pathways. Lastly, profiling age-dependent proteome changes in a caloric restriction model of yeast provided mechanistic insights into longevity, revealing minimal remodeling of energy-metabolic pathways, improved mitochondrial maintenance, ameliorated protein biogenesis, and decreased stress responses. Taken together, our study provides thousands of age-dependent molecular events that can be used to gain a holistic understanding of mechanisms of aging.

systems biology↗

bioRxiv2021

A modified fluctuation assay reveals a natural mutator phenotype that drives mutation spectrum variation within Saccharomyces cerevisiae

Although studies of Saccharomyces cerevisiae have provided many insights into mutagenesis and DNA repair, most of this work has focused on a few laboratory strains. Much less is known about the phenotypic effects of natural variation within S. cerevisiaes DNA repair pathways. Here, we use natural polymorphisms to detect historical mutation spectrum differences among several wild and domesticated S. cerevisiae strains. To determine whether these differences are likely caused by genetic mutation rate modifiers, we use a modified fluctuation assay with a CAN1 reporter to measure de novo mutation rates and spectra in 16 of the analyzed strains. We measure a 10-fold range of mutation rates and identify two strains with distinctive mutation spectra. These strains, known as AEQ and AAR, come from the panels "Mosaic beer" clade and share an enrichment for C>A mutations that is also observed in rare variation segregating throughout the genomes of several Mosaic beer and Mixed origin strains. Both AEQ and AAR are haploid derivatives of the diploid natural isolate CBS 1782, whose rare polymorphisms are enriched for C>A as well, suggesting that the underlying mutator allele is likely active in nature. We use a plasmid complementation test to show that AAR and AEQ share a mutator allele in the DNA repair gene OGG1, which excises 8-oxoguanine lesions that can cause C>A mutations if left unrepaired.

evolutionary biology↗

bioRxiv2020

Multiplexing Mutation Rate Assessment: Determining Pathogenicity of Msh2 Variants in S. cerevisiae

Despite the fundamental importance of mutation rate as a driving force in evolution and disease risk, common methods to assay mutation rate are time consuming and tedious. Established methods such as fluctuation tests and mutation accumulation experiments are low-throughput and often require significant optimization to ensure accuracy. We established a new method to determine the mutation rate of many strains simultaneously by tracking mutation events in a chemostat continuous culture device and applying deep sequencing to link mutations to alleles of a DNA-repair gene. We applied this method to assay the mutation rate of hundreds of Saccharomyces cerevisiae strains carrying mutations in the gene encoding Msh2, a DNA repair enzyme in the mismatch repair pathway (MMR). Loss-of-function (LOF) mutations in MSH2 are associated with hereditary non-polyposis colorectal cancer (HNPCC), an inherited disorder that increases risk for many different cancers. However, the vast majority of MSH2 variants found in human populations have insufficient evidence to be classified as either pathogenic or benign. We first benchmarked our method against Luria-Delbruck fluctuation tests using a collection of published MSH2 variants. Our pooled screen successfully identified previously-characterized non-functional alleles as high mutators. We then created an additional 185 human variants in the yeast ortholog, including both characterized and uncharacterized alleles curated from ClinVar and other clinical testing data. In a set of alleles of known pathogenicity, our assay recapitulated ClinVars classification; we then estimated pathogenicity for 157 variants classified as uncertain or conflicting reports of significance. This method is capable of studying the mutation rate of many microbial species and can be applied to problems ranging from the generation of high-fidelity polymerases to measuring the rate of antibiotic resistance emergence.

genetics↗

bioRxiv2019

Phenotypic and genotypic consequences of CRISPR/Cas9 editing of the replication origins in the rDNA of Saccharomyces cerevisiae

The complex structure and repetitive nature of eukaryotic ribosomal DNA (rDNA) is a challenge for genome assembly, and thus, the consequences of sequence variation in rDNA remain unexplored. However, renewed interest in the role that rDNA variation may play in diverse cellular functions, aside from ribosome production, highlights the need for a method that would permit genetic manipulation of the rDNA. Here, we describe a CRISPR/Cas9 based strategy to edit the rDNA locus in the budding yeast Saccharomyces cerevisiae. Using this approach, we modified the endogenous rDNA origin of replication in each repeat by deleting or replacing its consensus sequence. We characterized the transformants that have successfully modified their rDNA locus and propose a mechanism for how CRISPR/Cas9 mediated editing of the rDNA occurs. In addition, we carried out extended growth and life span experiments to investigate the long-term consequences that altering the rDNA origin of replication has on cellular health. We find that long-term growth of the edited clones results in faster growing suppressors that have acquired segmental aneusomy of the rDNA containing region of chr XII or aneuploidy of chromosomes XII, II, or IV. Furthermore, we find that all edited isolates suffer a reduced life span, irrespective of their levels of extrachromosomal rDNA circles. Our work demonstrates that it is possible to quickly, efficiently and homogeneously edit the rDNA locus via CRISPR/Cas9. It serves as a model for modifying other parts of the rDNA and, more generally, for editing other tandemly repeated sequences in higher eukaryotes.

genetics↗