Protracted Exposure to a Sub-background Radiation Environment Negatively Impacts the Anhydrobiotic Recovery of Desiccated Yeast Sentinels.

ABSTRACT: Experiments that examine the impacts of subnatural background radiation exposure provide a unique approach to studying the biological effects of low-dose radiation. These experiments often need to be conducted in deep underground laboratories in order to filter surface-level cosmic radiation. This presents some logistical challenges in experimental design and necessitates a model organism with minimal maintenance. As such, desiccated yeast ( Saccharomyces cerevisiae ) is an ideal model system for these investigations. This study aimed to determine the impact of prolonged sub-background radiation exposure in anhydrobiotic (desiccated) yeast at SNOLAB in Sudbury, Ontario, Canada. Two yeast strains were used: a normal wild type and an isogenic recombinational repair-deficient rad51 knockout strain ( rad51 Δ). Desiccated yeast samples were stored in the normal background surface control laboratory (68.0 nGy h -1 ) and in the sub-background environment within SNOLAB (10.1 nGy h -1 ) for up to 48 wk. Post-rehydration survival, growth rate, and metabolic activity were assessed at multiple time points. Survival in the sub-background environment was significantly reduced by a factor of 1.39 and 2.67 in the wild type and rad51 ∆ strains, respectively. Post-rehydration metabolic activity measured via alamarBlue reduction remained unchanged in the wild type strain but was 26% lower in the sub-background rad51 ∆ strain. These results demonstrate that removing natural background radiation negatively impacts the survival and metabolism of desiccated yeast, highlighting the potential importance of natural radiation exposure in maintaining homeostasis of living organisms.

BioSentinel: Validating Sensitivity of Yeast Biosensors to Deep Space Relevant Radiation.

With the imminent human exploration of deep space, it is more important than ever to understand the biological risks of deep space radiation exposure. The BioSentinel mission will be the first biological payload to study the effects of radiation beyond low Earth orbit in 50 years. This study is the last in a collection of articles about the BioSentinel biological CubeSat mission, where budding yeast cells will be used to investigate the response of a biological organism to long-term, low-dose deep space radiation. In this study, we define the methodology for detecting the biological response to space-like radiation using simulated deep space radiation and a metabolic indicator dye reduction assay. We show that there is a dose-dependent decrease in yeast cell growth and metabolism in response to space-like radiation, and this effect is significantly more pronounced in a strain of yeast that is deficient in DNA damage repair (rad51Δ) compared with a wild-type strain. Furthermore, we demonstrate the use of flight-like instrumentation after exposure to space-like ionizing radiation. Our findings will inform the development of novel and improved biosensors and technologies for future missions to deep space.

Toward sustainable space exploration: a roadmap for harnessing the power of microorganisms.

Finding sustainable approaches to achieve independence from terrestrial resources is of pivotal importance for the future of space exploration. This is relevant not only to establish viable space exploration beyond low Earth-orbit, but also for ethical considerations associated with the generation of space waste and the preservation of extra-terrestrial environments. Here we propose and highlight a series of microbial biotechnologies uniquely suited to establish sustainable processes for in situ resource utilization and loop-closure. Microbial biotechnologies research and development for space sustainability will be translatable to Earth applications, tackling terrestrial environmental issues, thereby supporting the United Nations Sustainable Development Goals.

BioSentinel: Long-Term Saccharomyces cerevisiae Preservation for a Deep Space Biosensor Mission.

The biological risks of the deep space environment must be elucidated to enable a new era of human exploration and scientific discovery beyond low earth orbit (LEO). There is a paucity of deep space biological missions that will inform us of the deleterious biological effects of prolonged exposure to the deep space environment. To safely undertake long-term missions to Mars and space habitation beyond LEO, we must first prove and optimize autonomous biosensors to query the deep space radiation environment. Such biosensors must contain organisms that can survive for extended periods with minimal life support technology and must function reliably with intermittent communication with Earth. NASA's BioSentinel mission, a nanosatellite containing the budding yeast Saccharomyces cerevisiae, is such a biosensor and one of the first biological missions beyond LEO in nearly half a century. It will help fill critical gaps in knowledge about the effects of uniquely composed, chronic, low-flux deep space radiation on biological systems and in particular will provide valuable insight into the DNA damage response to highly ionizing particles. Due to yeast's robustness and desiccation tolerance, it can survive for periods analogous to that of a human Mars mission. In this study, we discuss our optimization of conditions for long-term reagent storage and yeast survival under desiccation in preparation for the BioSentinel mission. We show that long-term yeast cell viability is maximized when cells are air-dried in trehalose solution and stored in a low-relative humidity and low-temperature environment and that dried yeast is sensitive to low doses of deep space-relevant ionizing radiation under these conditions. Our findings will inform the design and development of improved future long-term biological missions into deep space.

BioSentinel: A Biological CubeSat for Deep Space Exploration.

BioSentinel is the first biological CubeSat designed and developed for deep space. The main objectives of this NASA mission are to assess the effects of deep space radiation on biological systems and to engineer a CubeSat platform that can autonomously support and gather data from model organisms hundreds of thousands of kilometers from Earth. The articles in this special collection describe the extensive optimization of the biological payload system performed in preparation for this long-duration deep space mission. In this study, we briefly introduce BioSentinel and provide a glimpse into its technical and conceptual heritage by detailing the evolution of the science, subsystems, and capabilities of NASA's previous biological CubeSats. This introduction is not intended as an exhaustive review of CubeSat missions, but rather provides insight into the unique optimization parameters, science, and technology of those few that employ biological model systems.

BioSentinel: A Biofluidic Nanosatellite Monitoring Microbial Growth and Activity in Deep Space.

Small satellite technologies, particularly CubeSats, are enabling breakthrough research in space. Over the past 15 years, NASA Ames Research Center has developed and flown half a dozen biological CubeSats in low Earth orbit (LEO) to conduct space biology and astrobiology research investigating the effects of the space environment on microbiological organisms. These studies of the impacts of radiation and reduced gravity on cellular processes include dose-dependent interactions with antimicrobial drugs, measurements of gene expression and signaling, and assessment of radiation damage. BioSentinel, the newest addition to this series, will be the first deep space biological CubeSat, its heliocentric orbit extending far beyond the radiation-shielded environment of low Earth orbit. BioSentinel's 4U biosensing payload, the first living biology space experiment ever conducted beyond the Earth-Moon system, will use a microbial bioassay to assess repair of radiation-induced DNA damage in eukaryotic cells over a duration of 6-12 months. Part of a special collection of articles focused on BioSentinel and its science mission, this article describes the design, development, and testing of the biosensing payload's microfluidics and optical systems, highlighting improvements relative to previous CubeSat life-support and bioanalytical measurement technologies.

Enabling Clonal Analyses of Yeast in Outer Space by Encapsulation and Desiccation in Hollow Microparticles.

Studying microbes at the single-cell level in space can accelerate human space exploration both via the development of novel biotechnologies and via the understanding of cellular responses to space stressors and countermeasures. High-throughput technologies for screening natural and engineered cell populations can reveal cellular heterogeneity and identify high-performance cells. Here, we present a method to desiccate and preserve microbes in nanoliter-scale compartments, termed PicoShells, which are microparticles with a hollow inner cavity. In PicoShells, single cells are confined in an inner aqueous core by a porous hydrogel shell, allowing the diffusion of nutrients, wastes, and assay reagents for uninhibited cell growth and flexible assay protocols. Desiccated PicoShells offer analysis capabilities for single-cell derived colonies with a simple, low resource workflow, requiring only the addition of water to rehydrate hundreds of thousands of PicoShells and the single microbes encapsulated inside. Our desiccation method results in the recovery of desiccated microparticle morphology and porosity after a multi-week storage period and rehydration, with particle diameter and porosity metrics changing by less than 18% and 7%, respectively, compared to fresh microparticles. We also recorded the high viability of Saccharomyces cerevisiae yeast desiccated and rehydrated inside PicoShells, with only a 14% decrease in viability compared to non-desiccated yeast over 8.5 weeks, although we observed an 85% decrease in initial growth potential over the same duration. We show a proof-of-concept for a growth rate-based analysis of single-cell derived colonies in rehydrated PicoShells, where we identified 11% of the population that grows at an accelerated rate. Desiccated PicoShells thus provide a robust method for cell preservation before and during launch, promising a simple single-cell analysis method for studying heterogeneity in microbial populations in space.

Space Biology Research and Biosensor Technologies: Past, Present, and Future.

In light of future missions beyond low Earth orbit (LEO) and the potential establishment of bases on the Moon and Mars, the effects of the deep space environment on biology need to be examined in order to develop protective countermeasures. Although many biological experiments have been performed in space since the 1960s, most have occurred in LEO and for only short periods of time. These LEO missions have studied many biological phenomena in a variety of model organisms, and have utilized a broad range of technologies. However, given the constraints of the deep space environment, upcoming deep space biological missions will be largely limited to microbial organisms and plant seeds using miniaturized technologies. Small satellites such as CubeSats are capable of querying relevant space environments using novel, miniaturized instruments and biosensors. CubeSats also provide a low-cost alternative to larger, more complex missions, and require minimal crew support, if any. Several have been deployed in LEO, but the next iterations of biological CubeSats will travel beyond LEO. They will utilize biosensors that can better elucidate the effects of the space environment on biology, allowing humanity to return safely to deep space, venturing farther than ever before.

Characterization of the interaction between the Saccharomyces cerevisiae Rad51 recombinase and the DNA translocase Rdh54.

The Saccharomyces cerevisiae Rdh54 protein is a member of the Swi2/Snf2 family of DNA translocases required for meiotic and mitotic recombination and DNA repair. Rdh54 interacts with the general recombinases Rad51 and Dmc1 and promotes D-loop formation with either recombinase. Rdh54 also mediates the removal of Rad51 from undamaged chromatin in mitotic cells, which prevents formation of nonrecombinogenic complexes that can otherwise become toxic for cell growth. To determine which of the mitotic roles of Rdh54 are dependent on Rad51 complex formation, we finely mapped the Rad51 interaction domain in Rdh54, generated N-terminal truncation variants, and characterized their attributes biochemically and in cells. Here, we provide evidence suggesting that the N-terminal region of Rdh54 is not necessary for the response to the DNA-damaging agent methyl methanesulfonate. However, truncation variants missing 75-200 residues at the N terminus are sensitive to Rad51 overexpression. Interestingly, a hybrid protein containing the N-terminal region of Rad54, responsible for Rad51 interaction, fused to the Swi2/Snf2 core of Rdh54 is able to effectively complement the sensitivity to both methyl methanesulfonate and excess Rad51 in rdh54 null cells. Altogether, these results reveal a distinction between damage sensitivity and Rad51 removal with regard to Rdh54 interaction with Rad51.

Requirement of replication checkpoint protein kinases Mec1/Rad53 for postreplication repair in yeast.

UNLABELLED: DNA lesions in the template strand block the replication fork. In Saccharomyces cerevisiae, replication through DNA lesions occurs via a Rad6/Rad18-dependent pathway where lesions can be bypassed by the action of translesion synthesis (TLS) DNA polymerases η and ζ or by Rad5-mediated template switching. An alternative Rad6/Rad18-independent but Rad52-dependent template switching pathway can also restore the continuity of the replication fork. The Mec1/Rad53-dependent replication checkpoint plays a crucial role in the maintenance of stable and functional replication forks in yeast cells with DNA damage; however, it has remained unclear which of the lesion bypass processes requires the activation of replication checkpoint-mediated fork stabilization. Here we show that postreplication repair (PRR) of newly synthesized DNA in UV-damaged yeast cells is inhibited in the absence of Mec1 and Rad53 proteins. Since TLS remains functional in cells lacking these checkpoint kinases and since template switching by the Rad5 and Rad52 pathways provides the alternative means of lesion bypass and requires Mec1/Rad53, we infer that lesion bypass by the template switching pathways occurs in conjunction with the replication fork that has been stabilized at the lesion site by the action of Mec1/Rad53-mediated replication checkpoint. IMPORTANCE: Eukaryotic cells possess mechanisms called checkpoints that act to stop the cell cycle when DNA replication is halted by lesions in the template strand. Upon stalling of the ongoing replication at the lesion site, the recruitment of Mec1 and Rad53 kinases to the replication ensemble initiates the checkpoint wherein Mec1-mediated phosphorylation of Rad53 activates the pathway. A crucial role of replication checkpoint is to stabilize the replication fork by maintaining the association of DNA polymerases with the other replication components at the stall site. Our observations that Mec1 and Rad53 are required for lesion bypass by template switching have important implications for whether lesion bypass occurs in conjunction with the stalled replication ensemble or in gaps that could have been left behind the newly restarted forks. We discuss this important issue and suggest that lesion bypass in Saccharomyces cerevisiae cells occurs in conjunction with the stalled replication forks and not in gaps.

Analyses of the yeast Rad51 recombinase A265V mutant reveal different in vivo roles of Swi2-like factors.

The Saccharomyces cerevisiae Swi2-like factors Rad54 and Rdh54 play multifaceted roles in homologous recombination via their DNA translocase activity. Aside from promoting Rad51-mediated DNA strand invasion of a partner chromatid, Rad54 and Rdh54 can remove Rad51 from duplex DNA for intracellular recycling. Although the in vitro properties of the two proteins are similar, differences between the phenotypes of the null allele mutants suggest that they play different roles in vivo. Through the isolation of a novel RAD51 allele encoding a protein with reduced affinity for DNA, we provide evidence that Rad54 and Rdh54 have different in vivo interactions with Rad51. The mutant Rad51 forms a complex on duplex DNA that is more susceptible to dissociation by Rdh54. This Rad51 variant distinguishes the in vivo functions of Rad54 and Rdh54, leading to the conclusion that two translocases remove Rad51 from different substrates in vivo. Additionally, we show that a third Swi2-like factor, Uls1, contributes toward Rad51 clearance from chromatin in the absence of Rad54 and Rdh54, and define a hierarchy of action of the Swi2-like translocases for chromosome damage repair.

Role of DNA damage-induced replication checkpoint in promoting lesion bypass by translesion synthesis in yeast.

Unrepaired DNA lesions in the template strand block the replication fork. In yeast, Mec1 protein kinase-mediated replication checkpoint prevents the breakdown of replication forks and maintains viability in DNA-damaged cells going through the S phase. By ensuring that the replisome does not dissociate from the fork stalled at the lesion site, the replication checkpoint presumably coordinates the action of lesion bypass processes with the replisome. However, it has remained unclear as to which of the lesion bypass processes-translesion synthesis (TLS) and/or template switching-depend on the activation of the replication checkpoint. Here we determine if the Mec1 kinase and the subunits of the checkpoint clamp and the clamp loader are required for TLS. We show that proficient TLS can occur in the absence of these checkpoint proteins in nucleotide excision repair (NER)-proficient cells; however, in the absence of NER, checkpoint protein-mediated Rev1 phosphorylation contributes to increasing the proficiency of DNA polymerase zeta-dependent TLS.

Requirement of Nse1, a subunit of the Smc5-Smc6 complex, for Rad52-dependent postreplication repair of UV-damaged DNA in Saccharomyces cerevisiae.

In Saccharomyces cerevisiae, postreplication repair (PRR) of UV-damaged DNA occurs by a Rad6-Rad18- and an Mms2-Ubc13-Rad5-dependent pathway or by a Rad52-dependent pathway. The Rad5 DNA helicase activity is specialized for promoting replication fork regression and template switching; previously, we suggested a role for the Rad5-dependent PRR pathway when the lesion is located on the leading strand and a role for the Rad52 pathway when the lesion is located on the lagging strand. In this study, we present evidence for the requirement of Nse1, a subunit of the Smc5-Smc6 complex, in Rad52-dependent PRR, and our genetic analyses suggest a role for the Nse1 and Mms21 E3 ligase activities associated with this complex in this repair mode. We discuss the possible ways by which the Smc5-Smc6 complex, including its associated ubiquitin ligase and SUMO ligase activities, might contribute to the Rad52-dependent nonrecombinational and recombinational modes of PRR.

Chromosomal aberrations in peripheral lymphocytes from male native miners working in the Peruvian Andes

We analyzed the frequency of chromosomal aberrations in peripheral lymphocytes from underground miners from the Casapalca (n = 8, mean age = 45 y, range = 36 y to 55 y) and Bellavista (n = 8, mean age = 28 y, range 23 y to 34 y) high-altitude mining camps in the Peruvian Andes. This population was occupationally exposed to heavy metals such as lead and zinc as well as diesel emission particles, organic solvents and mine dust. The control groups consisted of individuals from a high altitude farming community in the Peruvian village of Tinco (n = 8, mean age = 37 y, range = 25 y to 52 y) and the sea level city of Lima (n = 14, mean age = 26 y, range = 20 y to 35 y). All individuals were male native Peruvians. A significantly higher incidence (1.88 percent, p < 0.05) of chromosomal aberrations (chromatid deletions and breaks, chromosome breaks and acentric fragments) were detected in lymphocytes from miners at the Casapalca camp as compared to miners from the Bellavista camp (0.5 percent, chromatid deletions and acentric fragments only) and the Lima sea level (0.07 percent, chromatid deletions only) and Tinco high altitude (no aberrations) controls. These results suggest that male native Peruvians occupationally exposed to underground mining activity have an increased frequency of chromosomal aberrations, which could be related to both age and exposure time. The increased chromosomal damage observed in the mining populations studied may be attributable to the complex mixture of genotoxic agents to which the miners may have been exposed.