Mitochondria-encoded genes contribute to evolution of heat and cold tolerance in yeast

Genetic analysis of phenotypic differences between species is typically limited to interfertile species. Here, we conducted a genome-wide noncomplementation screen to identify genes that contribute to a major difference in thermal growth profile between two reproductively isolated yeast species, Saccharomyces cerevisiae and Saccharomyces uvarum. The screen identified only a single nuclear-encoded gene with a moderate effect on heat tolerance, but, in contrast, revealed a large effect of mitochondrial DNA (mitotype) on both heat and cold tolerance. Recombinant mitotypes indicate that multiple genes contribute to thermal divergence, and we show that protein divergence in COX1 affects both heat and cold tolerance. Our results point to the yeast mitochondrial genome as an evolutionary hotspot for thermal divergence.This work was supported by the NIH (grant GM080669) to J.C.F. Additional support to C.T.H. was provided by the USDA National Institute of Food and Agriculture (Hatch project 1003258), the National Science Foundation (DEB-1253634), and the DOE Great Lakes Bioenergy Research Center (DOE BER Office of Science DE-SC0018409 and DE-FC02-07ER64494 to T. J. Donohue). C.T.H. is a Pew Scholar in the Biomedical Sciences and a Vilas Faculty Early Career Investigator, supported by the Pew Charitable Trusts and the Vilas Trust Estate, respectively. D.P. is a Marie Sklodowska-Curie fellow of the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 747775).Peer reviewe

The global retinoblastoma outcome study : a prospective, cluster-based analysis of 4064 patients from 149 countries

DATA SHARING : The study data will become available online once all analyses are complete.BACKGROUND : Retinoblastoma is the most common intraocular cancer worldwide. There is some evidence to suggest that major differences exist in treatment outcomes for children with retinoblastoma from different regions, but these differences have not been assessed on a global scale. We aimed to report 3-year outcomes for children with retinoblastoma globally and to investigate factors associated with survival. METHODS : We did a prospective cluster-based analysis of treatment-naive patients with retinoblastoma who were diagnosed between Jan 1, 2017, and Dec 31, 2017, then treated and followed up for 3 years. Patients were recruited from 260 specialised treatment centres worldwide. Data were obtained from participating centres on primary and additional treatments, duration of follow-up, metastasis, eye globe salvage, and survival outcome. We analysed time to death and time to enucleation with Cox regression models. FINDINGS : The cohort included 4064 children from 149 countries. The median age at diagnosis was 23·2 months (IQR 11·0–36·5). Extraocular tumour spread (cT4 of the cTNMH classification) at diagnosis was reported in five (0·8%) of 636 children from high-income countries, 55 (5·4%) of 1027 children from upper-middle-income countries, 342 (19·7%) of 1738 children from lower-middle-income countries, and 196 (42·9%) of 457 children from low-income countries. Enucleation surgery was available for all children and intravenous chemotherapy was available for 4014 (98·8%) of 4064 children. The 3-year survival rate was 99·5% (95% CI 98·8–100·0) for children from high-income countries, 91·2% (89·5–93·0) for children from upper-middle-income countries, 80·3% (78·3–82·3) for children from lower-middle-income countries, and 57·3% (52·1-63·0) for children from low-income countries. On analysis, independent factors for worse survival were residence in low-income countries compared to high-income countries (hazard ratio 16·67; 95% CI 4·76–50·00), cT4 advanced tumour compared to cT1 (8·98; 4·44–18·18), and older age at diagnosis in children up to 3 years (1·38 per year; 1·23–1·56). For children aged 3–7 years, the mortality risk decreased slightly (p=0·0104 for the change in slope). INTERPRETATION : This study, estimated to include approximately half of all new retinoblastoma cases worldwide in 2017, shows profound inequity in survival of children depending on the national income level of their country of residence. In high-income countries, death from retinoblastoma is rare, whereas in low-income countries estimated 3-year survival is just over 50%. Although essential treatments are available in nearly all countries, early diagnosis and treatment in low-income countries are key to improving survival outcomes.The Queen Elizabeth Diamond Jubilee Trust and the Wellcome Trust.https://www.thelancet.com/journals/langlo/homeam2023Paediatrics and Child Healt

Analysis of Repeat-Mediated Deletions in the Mitochondrial Genome of Saccharomyces cerevisiae

Mitochondrial DNA deletions and point mutations accumulate in an age-dependent manner in mammals. The mitochondrial genome in aging humans often displays a 4977-bp deletion flanked by short direct repeats. Additionally, direct repeats flank two-thirds of the reported mitochondrial DNA deletions. The mechanism by which these deletions arise is unknown, but direct-repeat-mediated deletions involving polymerase slippage, homologous recombination, and nonhomologous end joining have been proposed. We have developed a genetic reporter to measure the rate at which direct-repeat-mediated deletions arise in the mitochondrial genome of Saccharomyces cerevisiae. Here we analyze the effect of repeat size and heterology between repeats on the rate of deletions. We find that the dependence on homology for repeat-mediated deletions is linear down to 33 bp. Heterology between repeats does not affect the deletion rate substantially. Analysis of recombination products suggests that the deletions are produced by at least two different pathways, one that generates only deletions and one that appears to generate both deletions and reciprocal products of recombination. We discuss how this reporter may be used to identify the proteins in yeast that have an impact on the generation of direct-repeat-mediated deletions

Double strand break repair and genetic analyses of Rad27p in the mitochondria of Saccharomyces cerevisiae

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2015.Mitochondria contain their own genetic material, which is essential for energy production in eukaryotes. Similar to nuclear DNA, mitochondrial DNA (mtDNA) is also under constant mutagenic stress and the accumulation of mutations in this genome has been associated with a wide range of metabolic and degenerative diseases, certain types of cancer, and aging. Therefore, maintaining the integrity of this genetic material is crucial for cellular homeostasis. Several DNA repair pathways including homologous recombination (HR) have been identified in this organelle but the mechanistic details and the protein players involved in these processes have remained elusive. To investigate HR mechanisms and DNA repair proteins in mitochondria, we engineered a system to induce a specific double strand break (DSB) in the mtDNA of Saccharomyces cerevisiae and study its repair. Using this system, we have determined that induced DSBs at a locus flanked by repeat sequences are repaired by an erroneous HR mechanism, which results in deletion of the region intervening the repeats. Furthermore, DSB repair involves processing of DNA ends at the break site, which produces single stranded recombinogenic intermediates. Our results show that generation of deletions upon DSB induction and DNA end resection requires the function of Rad27p and Nuc1p. Both these proteins localize to the nucleus as well as mitochondria but their precise function in the latter is poorly understood. To determine the roles of the multifunctional Rad27p in mitochondria, we examined the mutagenic effects of various nuclease deficient alleles of this protein in this cellular compartment. Our results demonstrate that Rad27p functions in more than one pathway in mitochondria, using its multiple enzymatic activities. Furthermore, based on our findings, we conclude that the yeast Rad27p uses a cryptic internal mitochondrial-targeting signal, and an import mechanism that is likely different from its mammalian homolog, FEN1

The role of homologous recombination in the maintenance and repair of the mitochondrial genome

Thesis (Ph. D.)--University of Rochester. Department of Biology, 2017.Mitochondria are dynamic and multifunctional organelles. While the mitochondrion is traditionally thought of as the “power house” of the cell, it has many functions beyond this, including roles in the biosynthesis of amino acids and lipids, signalling, and calcium homeostasis. Mitochondria also contain their own genome that must be replicated and repaired faithfully. Mutations and deletions in the mitochondrial genome are associated with a number of human diseases and syndromes. Homologous recombination is an essential process in the nucleus that facilitates DNA replication, repair, and chromosome segregation. The contribution of homologous recombination to mitochondrial DNA (mtDNA) metabolism is less well understood in comparison. Using our direct repeat mediated deletion assay and an optimized mitochondrial induced double-strand break assay, I have characterized the role of Rad51p, Rad52p, Rad59p, Cce1p, and Irc3p in mitochondrial homologous recombination. Based on our findings, we have concluded that both Rad51p and Rad59p are localized to the matrix of the mitochondria, and that Rad51p and Irc3p bind directly to mtDNA. We find that loss of each of these three proteins significantly decreases the rate of spontaneous deletion events and the loss of Rad51p and Rad59p impairs the repair of induced mtDNA DSBs. Cce1p prevents spontaneous deletions from occurring under certain growth conditions, suggesting that the overall metabolic state of the cell can influence the stability of the mtDNA. Irc3p is critical to the overall stability of the mitochondrial genome. Without Irc3p, the frequency of spontaneous deletions and respiration loss is extremely high, which suggests that Irc3p may play a critical role in several aspects of mitochondrial homologous recombination. Taken together these studies have revealed that homologous recombination proteins function in diverse pathways that all impact mtDNA metabolism

DNA damage tolerance and repair pathways in the mitochondrial compartment

Thesis (Ph. D.)--University of Rochester. Dept. of Biology, 2011.Mitochondria are essential organelles in nearly all eukaryotic cells. Due to their crucial function in respiration, mitochondria are frequently referred to as the powerhouses of the cell. These highly dynamic structures contain their own DNA genome, and the stability of this genome is fundamental in maintaining normal cellular function. Mutations in the human mitochondrial genome have been implicated in many diseases, including neuromuscular disorders, heart disease, and diabetes. Moreover, the accumulation of mutations in the mitochondrial genome has been shown to contribute to aging and aging-related disorders. Mutations in DNA occur as a result of errors in DNA synthesis and repair processes acting on damaged DNA. Although nuclear processes responsible for DNA replication and repair are well studied, pathways involved in mitochondrial DNA replication and repair are comparatively poorly understood. The work presented here describes the identification and characterization of mechanisms involved in DNA damage tolerance and repair in the mitochondrial compartment. In this study, I performed a screen of the Saccharomyces cerevisiae deletion collection to identify novel candidate genes involved in maintaining mitochondrial DNA integrity. Results from this analysis revealed that several proteins required for nuclear nucleotide excision repair function in mitochondrial DNA mutation avoidance after UV-induced damage. Additionally, I have demonstrated that a highly conserved nuclear protein FEN1, and its yeast homolog Rad27p, localize to the mitochondrial compartment in yeast and mice. I have completed a comprehensive genetic analysis of RAD27 mutants and describe the functional relevance in mitochondrial base excision repair as well as mitochondrial DNA maintenance. Furthermore, I have characterized the impact of translesion DNA polymerases on mitochondrial DNA stability and revealed a novel, mitochondrial-specific mechanism of mutagenesis. Lastly, I have critically examined the pathways involved in mitochondrial DNA double-strand break repair. Using novel reporter constructs, I implicate roles for the nuclear double-strand break repair proteins in generating mitochondrial DNA deletions. Moreover, I have generated a system to induce a single, specific mitochondrial double-strand break allowing for the analysis of the kinetics of this type of repair. In summary, these studies have identified novel mechanisms of maintaining mitochondrial DNA integrity, as well as, developed our current understanding of mitochondrial DNA mutagenesis