Abstract
We investigate whether a protein found in tardigrades called Damage suppressor, or Dsup, can help cells restart stalled replication forks in human cells. Tardigrades compose a phylum of microscopic animals known for their ability to survive extreme environmental conditions, and experiments have shown that Dsup prevents damage from accumulating in DNA (Hashimoto et al. 2016). Excessive stalled replication forks can result in DNA degradation and cell death (Ceccaldi et al. 2016). Human Fanconi anemia protein FANCD2 is required to restart stalled forks (Chaudhury et al. 2013). We have expressed Tardigrade Dsup protein in human FANCD2 deficient cells to determine the role of Dsup on DNA replication restart and cellular viability in response to replication inhibitor. Our results strongly support the hypothesis that Dsup is a novel DNA repair protein which helps stalled replication forks restart.
Introduction
Life’s not easy, not even at the microscopic level. Every day, our cells are bombarded by radiation and metabolic toxins that threaten to tear apart our DNA – damage that could either kill the cells, or corrupt them until they turn cancerous. Interstrand crosslinks are particularly devastating, as they prevent the separation of DNA strands, hindering replication. If left uncorrected, as few as twenty interstrand crosslinks can kill a cell (Huang and Li 2013).
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Fortunately, our cells are not helpless, but instead have evolved sophisticated repair mechanisms to survive. For example, the Fanconi anemia pathway consists of about twenty core proteins working together to repair interstrand crosslinks. This pathway is so essential that people missing one or more of the Fanconi anemia proteins may be vulnerable to bone marrow failure, developmental abnormalities, and cancer (Ceccaldi et al. 2016).
Interestingly, other organisms may have evolved yet more ways to protect their DNA from damage. The tardigrades, a phylum over 1000 species strong, are renowned for their durability. Some species can withstand temperatures close to absolute zero (-273 degrees C), radiation exposure, or temporary excursions in outer space. Part of their resilience might be attributed to a special protein found only in tardigrades, called the Damage suppressor, or Dsup (Hashimoto and Kunieda 2017).
Hashimoto and colleagues (2016) demonstrated that Dsup helps protect DNA from both single-stranded and double-stranded DNA breaks. When they genetically engineered human cells to express Dsup, they found that the cells were more resistant to X-ray and hydrogen peroxide exposure. Cultures of cells containing Dsup proliferated more rapidly than cells lacking Dsup, suggesting improved viability. Hashimoto and colleagues propose that Dsup may physically shield the DNA, preventing any damage from occurring at all. Other researchers suggest that Dsup may assist with repair (Nikitaki et al. 2018). The precise role (or roles) of Dsup within the cell is unclear.
We sought to discover if Dsup could assist in DNA repair and replication by comparing its performance to the Fanconi anemia protein FANCD2. For our experiment, we used four different cells: some expressing FANCD2, some expressing Dsup, some expressing neither protein, and the rest expressing both. We exposed the cells to aphidicolin, a toxin that temporarily inhibits DNA polymerase and causes replication forks to stall (Nayeri 2014). FANCD2 is essential for restarting stalled replication forks and preventing nascent DNA degradation (Chaudhury et al. 2013). Usually, cells with FANCD2 can recover after exposure to aphidicolin. If Dsup can likewise help restart replication forks, then cells lacking FANCD2 but possessing Dsup should also be able to continue replicating after treatment with aphidicolin.
Methods
We conducted three different experiments: DNA fiber analysis, colony formation assays, and immunoprecipitation analysis. For all of these experiments, we compared how four different types of cells coped with aphidicolin.
Cells: The Fanconi Anemia Cell Repository, Oregon provided the human patient fibroblast cells used in our experiment. PD20+D2 cells were used as wild-type and contained the functional FANCD2 gene. PD20 cells, on the other hand were FANCD2 deficient. We transiently transfected both types of cells using vectors provided by AddGene and a lipofectamine reagent. Half of the cells were transfected with an empty vector while the other half were transfected with a vector that contained the functional full-length Tardigrade Dsup gene. As the cells were growing, they were fed a media of DMEM supplemented with 10% FBS and antibiotics. We performed our experiments after 72 hours of transfection.
DNA Fiber Analysis: For this experiment, we labeled our cells by treating them with 50 uM IdU for 30 min. We then exposed half the cells to 30 μM aphidicolin for 3 hours, leaving the other half untreated to serve as controls. Next, we treated the cells with 50 uM CldU for 30 min. We fixed the cells and spread the fibers on glass slides coated with silane. Finally, we treated the slides with 1:1000 Rat anti-BrdU clone BU1/75 antibody (AbD Serotec), Alexa Fluor 488 goat ani-rat secondary antibody, mouse anti-BrdU clone B44 antibody (Becton Dickinson) and Alexa Flour 546 goat anti-mouse secondary antibody.
Once our slides were prepared, we used Deltavision microscope (University of Minnesota, Twin Cities) to capture images of the DNA strands. IdU-labeled DNA strands are Red in color whereas CldU-labeled tracts are green. We then determined the proportion of multicolored (Red-Green; restarted forks) strands to red-only (stalled forks) strands to figure out what fraction was able to restart.
Colony Formation Assays: We exposed the cells for 3 hours to different concentrations of aphidicolin: 30 μM, 3 μM, 0.3 μM, and 0 μM. We cultured the cells for ~ 7 days, and afterwards used crystal violet solution to stain the cells. Then, we used a compound light microscope to approximate the number of colonies for each sample.
Immunoprecipitation: We treated half our cells with 30 μM aphidicolin for 3 hours, and left the remainder alone. We then captured proteins with FANCD2 antibody beads in half our samples and IgG antibody beads in the other half.
DNA Fiber Analysis
As depicted in the table and graph above, the results of the first trial of our fiber analysis experiment show differences in the proportion of multicolored strands to red strands depending on both the genotype of the cells and whether or not they were exposed to aphidicolin. The multicolored strands show that cells were able to replicate their DNA during both the stages of IdU and CldU exposure. In contrast, the red strands indicate stalled forks. Unsurprisingly, the cells that were not treated with aphidicolin had more multicolored strands than the cells that were treated with aphidicolin. For the untreated condition, 91% of strands were multicolored in the cells that possessed both the Dsup and FANCD2 genes, 90% were multicolored in cells that had either the FANCD2 or the Dsup gene, and 83% were multicolored in the cells that had neither the FANCD2 nor the Dsup gene. As for the treated condition, 74% of strands were multicolored in cells that had both the Dsup and FANCD2 genes, 58% were multicolored in cells that had only the FANCD2 gene, 37% were multicolored in cells that had only the Dsup gene, and 6% were multicolored in cells that had neither the Dsup gene nor the FANCD2 gene.
While the proportions of multicolored strands in our first trial varied dramatically depending on the cell type and experimental condition, we will need the images for the other two trials to establish whether the differences are statistically significant. The results for these final two trials are still pending.
Colony Formation Assays
The results of our colony formation assays are inconclusive. For the first trial, the number of colonies declined for all groups as the amount of aphidicolin increased. On average, the cells with only the Dsup gene grew the most colonies, while cells with only the FANCD2 gene grew the fewest colonies. Cells that had both the Dsup and the FANCD2 gene grew more colonies than the cells that had neither. However, the differences between groups were small overall.
Unfortunately, during the second trial, the cells had overpopulated their dishes and started to die off, compromising the results. Therefore, we were unable to verify our observations from the first trial.
Discussion
Results of our DNA fiber analysis are promising. As expected, wild type cells were able to restart their replications after exposure to aphidicolin while FANCD2 deficient cells were not. Interestingly, expression of tardigrade Dsup promoted replication restart in FANCD2-deficient cells strongly suggesting the role of Dsup during replication stress.
Similarly, more trials of the colony formation assays will also need to be done before we can determine how the presence of the Dsup protein impacts how well cells can grow after exposure to different amounts of aphidicolin. While the first trial might indicate that Dsup helps the cells grow, the differences in colony numbers between cell groups were relatively small, and random variation may have clouded any true underlying patterns. Indeed, since the results of the first trial suggest that cells with FANCD2 develop fewer colonies than cells without, we might be wary developing any conclusions just yet. Only about 6.6% of each petri dish was observed under the microscope as we collected data, so a larger sample size could have improved the validity of our colony counts. Furthermore, additional trials would have enabled greater confidence.
Our experiments do not exclude the possibility that Dsup performs multiple roles within the cell, nor do they completely elucidate the precise mechanisms of the protein’s functions. More experiments will need to be conducted if we are to understand how proteins like Dsup can help organisms survive a challenging world.
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