Adaptive evolution of a melanized fungus reveals robust augmentation of radiation resistance by abrogating non‐homologous end‐joining

Abstract

Fungi have been observed to exhibit resistance to high levels of ionizing radiation despite sharing most DNA repair mechanisms with other eukaryotes. Radioresistance, in fact, is such a common feature in fungi that it is difficult to identify species that exhibit widely different radiosensitivities, which in turn has hampered the identification of genetic elements responsible for this resistance phenotype. Due to the inherent mutagenic properties of radiation exposure, however, this can be addressed through adaptive laboratory evolution for increased ionizing radiation resistance. Here, using the black yeast Exophiala dermatitidis, we demonstrate that resistance to γ-radiation can be greatly increased through repeated rounds of irradiation and outgrowth. Moreover, we find that the small genome size of fungi situates them as a relatively simple functional genomics platform for identification of mutations associated with ionizing radiation resistance. This enabled the identification of genetic mutations in genes encoding proteins with a broad range of functions from 10 evolved strains. Specifically, we find that greatly increased resistance to γ-radiation is achieved in E. dermatitidis through disruption of the non-homologous end-joining pathway, with three individual evolutionary paths converging to abolish this DNA repair process. This result suggests that non-homologous end-joining, even in haploid cells where homologous chromosomes are not present during much of the cell cycle, is an impediment to repair of radiation-induced lesions in this organism, and that the relative levels of homologous and nonhomologous repair in a given fungal species may play a major role in its radiation resistance.

“Genetic scissors” CRISPR/Cas9 genome editing cutting-edge biocarrier technology for bone and cartilage repair

“…The first one is nonhomologous end joining (NHEJ), which urgently links the broken DNA double strands like spontaneous “SOS” (“save our souls” signal) repair in living organisms. However, this repair method is stochastic and prone to insertion or deletion mutations that can damage the target gene [ 26 ]. The other one is homology-directed repair (HDR), which is a small fragment of DNA with the same sequence at both ends and the broken sequence can be homologously recombined with the broken gene, thus completing the exact recombination of the gene [ 27 ].…”

Phenotypic Characterization and Comparative Genomics of the Melanin-Producing Yeast Exophiala lecanii-corni Reveals a Distinct Stress Tolerance Profile and Reduced Ribosomal Genetic Content

“…Phenotypic characterization coupled with comparative genomic analysis can strengthen our understanding of the genetic underpinnings that define variation among black yeasts. Our research group has previously conducted numerous studies into the black yeast E. dermatitidis that have advanced our understanding of the molecular basis of its extremophilic characteristics, as well as the impact of melanin on such processes [6,[27][28][29][30]. In a previous study, we observed that although E. dermatitidis produces substantially less melanin than E. lecanii-corni, E. dermatitidis is far more resistant to high-dose acute ionizing radiation [6].…”

Data_Sheet_1.docx

“…Another insight we gleaned from the current dataset are the possible roles of autophagy and protein catabolism in recovery from particle irradiation. While DNA is considered to be the prime target for IR-induced damage, and genome instability the major cause of IR-induced cell death, protein oxidation is a known effect of IR-induced free-radical production [for detailed discussions on this topic, see Cabiscol et al (2000), Braunstein et al (2009), Daly (2012), Höhn et al (2013), Radman (2016), Romsdahl et al (2020]. Effects of IR on lipids, including changes in the levels of phospholipids and sterols in the plasma membrane, disruption of regulated membrane transport, and general lipid peroxidation are also known, though to a lesser extent (Stark, 1991;Kiang et al, 2012;Laiakis et al, 2014).…”

“…These organisms are indeed good models for this work -they have small, easily manipulated genomes and can be grown and irradiated easily (Loftus et al, 2005;Dadachova et al, 2007;Zakrzewska et al, 2011;Chen et al, 2014;Jung et al, 2016). Significant findings from studies in these organisms include the stimulation of growth of some fungi by low dose IR (Dadachova et al, 2007Dadachova and Casadevall, 2008); the possibility that melanin in the cell walls of certain fungi may protect against IR damage Pacelli et al, 2017a); the high IR resistance of organisms such as Ustilago maydis (Holliday, 1975;Lee and Yarranton, 1982;Holloman et al, 2007;Milisavljevic et al, 2018), Cryptococcus neoformans (Shuryak et al, 2014;Jung et al, 2016;Pacelli et al, 2017a;Schultzhaus et al, 2019), Cryomyces antarcticus (Pacelli et al, 2017b), and Exophiala dermatitidis (Romsdahl et al, 2020;; and the discovery of novel IR resistance-associated proteins through transcriptomics and targeted mutagenesis (Jung et al, 2016;Schultzhaus et al, 2019;.…”

“…We are interested in the factors that mediate the response to IR exposure in fungi, and have previously characterized how melanin affects the response of E. dermatitidis to γ-IR, because it is an intrinsically melanized fungus that is highly radioresistant (Robertson et al, 2012;Romsdahl et al, 2020;. Acute and chronic γ-IR exposures induced dramatic transcriptomic responses in E. dermatitidis that allowed us to identify previously uncharacterized proteins important for its resistance, but nothing is currently known about how these results relate to the effects that ionizing particles have on this organism.…”