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Browsing by Author "Waneka, Gus, author"

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    The tempo and mode of angiosperm mitochondrial genome divergence inferred from intraspecific variation in Arabidopsis thaliana
    (Colorado State University. Libraries, 2020-01-21) Wu, Zhiqiang, author; Waneka, Gus, author; Sloan, Daniel B., author; Genetics Society of America, publisher
    The mechanisms of sequence divergence in angiosperm mitochondrial genomes have long been enigmatic. In particular, it is difficult to reconcile the rapid divergence of intergenic regions that can make non-coding sequences almost unrecognizable even among close relatives with the unusually high levels of sequence conservation found in genic regions. It has been hypothesized that different mutation/repair mechanisms act on genic and intergenic sequences or alternatively that mutational input is relatively constant but that selection has strikingly different effects on these respective regions. To test these alternative possibilities, we analyzed mtDNA divergence within Arabidopsis thaliana, including variants from the 1001 Genomes Project and changes accrued in published mutation accumulation (MA) lines. We found that base-substitution frequencies are relatively similar for intergenic regions and synonymous sites in coding regions, whereas indel and nonsynonymous substitutions rates are greatly depressed in coding regions, supporting a conventional model in which mutation/repair mechanisms are consistent throughout the genome but differentially filtered by selection. Most types of sequence and structural changes were undetectable in 10-generation MA lines, but we found significant shifts in relative copy number across mtDNA regions for lines grown under stressed vs. benign conditions. We confirmed quantitative variation in copy number across the A. thaliana mitogenome using both whole-genome sequencing and droplet digital PCR, further undermining the classic but oversimplified model of a circular angiosperm mtDNA structure. Our results suggest that copy number variation is one of the most fluid features of angiosperm mitochondrial genomes.
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    Uncovering mechanisms driving variation in mutation rates in organellar and nuclear genomes
    (Colorado State University. Libraries, 2024) Waneka, Gus, author; Sloan, Daniel B., advisor; Lockridge Mueller, Rachel, committee member; Argueso, Juan Lucas, committee member; Stenglein, Mark, committee member
    Mutations are changes to DNA sequences which drive evolution by supplying raw genetic variation for natural selection to act upon. At the same time, mutations tend to have negative fitness consequences and are the source of genetic diseases. Such costs and benefits of mutation create opposing forces of selection on mutation rate modifiers, which are alleles (typically in DNA repair genes) responsible for increases or decreases to mutation rates. Essentially all eukaryotes possess at least two genomes: the nuclear genome (nucDNA) and the endosymbiotically derived mitochondrial genome (mtDNA). Photosynthetic plants and algae additionally possess endosymbiotically derived plastid genomes (cpDNAs). Together, the mtDNA and cpDNA are referred to as organellar genomes. Chapter 1 of this dissertation provides a framework for understanding how DNA repair machinery and mutation rates have evolved in complex eukaryotic cells. Chapters 2 and 3 focus on specific repair pathways active in organellar genomes. Finally, Chapter 4 shifts focus to understand how environmental perturbations in expression level impact mutation in plant nuclear genomes. Repair of organellar genomes is conducted by nuclear-encoded genes that are translated in the cytosol and targeted to the organelles. In terms of evolutionary history, organellar repair machinery is a mosaic network of bacterial-like repair genes, which came into the cell with the organelles, and nuclear repair genes that have been co-opted for organellar function. In some cases, repair proteins are targeted to both the nucDNA and mtDNA (and/or cpDNA in plants) to perform similar functions. This is the case as for many base excision repair (BER) proteins, which identify and remove of chemically damaged bases. In contrast, organellar repair arsenals are thought to lack canonical mismatch-repair (MMR) and nucleotide excision repair (NER), which are both important repair pathways in nuclear genomes. Instead, the diverse eukaryotic lineages have adopted unique strategies for organellar genome maintenance. As a result, there is a tremendous diversity in mtDNA mutation rates, which show over a 4000-fold variation across eukaryotes. Interestingly, much of this variation is driven by the extremely low point mutation rates plant mtDNAs. In addition, plant organellar genomes are more recombinationally active and plant mtDNAs are structurally unstable compared to the mtDNAs of other eukaryotes. Chapter 2 of this dissertation explores the mechanistic basis of low point mutation rates and recombination-mediated repair in plant organellar genomes. We performed high-fidelity Duplex Sequencing on a panel of Arabidopsis thaliana lines lacking specific organellar genome repair genes. We report large point mutation increases in mutant lines lacking MSH1, a mutS homolog that has been proposed to induce double-stranded breaks at the site of DNA mismatches, effectively shuttling such lesions into homologous recombination (HR) pathways that play important roles in plant organellar genome replication and repair. We see smaller point mutation increases in other mutant lines lacking RADA, RECA1 and RECA3. In addition, we generated long-read Oxford Nanopore sequencing to characterize repeat-mediated recombination in several of the mutants in the panel. Our findings provide valuable insights into the mechanisms driving the fascinating patterns of organellar genome evolution in land plants. The aforementioned lack of NER in organellar genomes is surprising given the importance of NER as a 'catchall' for repair of a variety of bulky DNA lesions in nuclear genomes. Chapter 3 focuses in on the fate of photodamage (an important type of bulky DNA damage) in organellar DNA. To do so, we leverage publicly available XR-seq datasets, which were generated to quantify and map active NER excision products in nuclear genomes following UV exposure. The taxonomic scope of chapter is expanded from plants to also include fungi (the brewer's yeast Saccharomyces cerevisiae) and animals (the model fruit fly Drosophila melanogaster). We find that mtDNA-derived XR-seq reads in A. thaliana and S. cerevisiae have distinct and repeatable patterns in terms of length and internal positioning of pyrimidine dimers (known targets of photodamage formation). These data mirror established patterns of NER-derived reads originating from the nuclear genomes, raising the exciting possibility that NER-like repair pathways may exist in for repair of photodamage in organellar genomes. The focus of chapter 4 shifts to understanding how environmental changes impact mutation in plant nuclear genomes. The textbook view of mutation and adaptation is that mutations occur randomly with respect to their environment-specific fitness consequences. However, this view of random mutation is challenged as evidence increasingly establishes a correlation between increased expression and decreased mutation via the coordination of transcription and DNA repair machinery at the molecular level. As a result of this correlation, intragenomic mutation rates likely vary with changing environments given that expression levels are environmentally labile. Therefore, certain genes may be predisposed to higher or lower mutation rates depending on the environment, though the magnitude and importance of this effect remains largely untested. A technical challenge in addressing these questions is that large scale plant mutation datasets are time and resource intensive to generate. A recent plant study relied on low frequency somatic calls from Illumina based shotgun libraries to generate a large number of mutations, but others report that most of these inferred mutations are sequencing errors. To overcome these challenges, we took a novel approach to measuring somatic mutations by using Duplex Sequencing to quantify mutations in targeted regions of the A. thaliana nucDNA. We identified a set of differentially expressed genes in plants grown at different temperatures, which we then targeted for mutation detection using hybrid capture. In addition to wild type (WT) lines, we also studied mutant lines deficient in BER and MMR to test if either of these pathways are responsible for the correlation between expression and mutation in plants. We found large point mutation increases in the MMR mutants compared to WT plants, which displayed surprisingly few mutations at either temperature. Though the small number of WT mutations precluded a meaningful comparison of expression and mutation in a WT background, this result if nonetheless valuable for establishing that the true frequency of somatic mutations in plants is indeed very low suggesting that previous estimates likely conflated Illumina based sequencing artifacts with mutations. Mutation rates vary by over three orders of magnitude across the tree of life. Much of this variation is captured in mitochondrial mutation rates. The chapters of this dissertation provide valuable insights into the molecular processes that drive mutation rate variation in eukaryotic genomes. Such mechanistic understandings are critical for advancing the broader field of mutation rate evolution.