The Tardigrade Damage Suppressor Protein Modulates Transcription Factor and DNA Repair Genes in Human Cells Treated with Hydroxyl Radicals and UV-C

Abstract

The Ramazzottius varieornatus tardigrade is an extremotolerant terrestrial invertebrate with a length of 0.1–1.0 mm. These small animals show an extraordinary tolerance to extreme conditions such as high pressure, irradiation, chemicals and dehydration. These abilities are linked to a recently discovered damage suppressor protein (Dsup). Dsup is a nucleosome-binding protein that avoids DNA damage after X-ray and oxidative stress exposure without impairing cell life in Dsup-transfected animal and plant cells. The exact “protective” role of this protein is still under study. In human cells, we confirmed that Dsup confers resistance to UV-C and H2O2 exposure compared to untransfected cells. A different transcription factor activation was also observed. In addition, a different expression of endogenous genes involved in apoptosis, cell survival and DNA repair was found in Dsup+ cells after H2O2 and UV-C. In UV-C exposed cells, Dsup efficiently upregulates DNA damage repair genes, while H2O2 treatment only marginally involves the activation of pathways responsible for DNA repair in Dsup+ cells. These data are in agreement with the idea of a direct protective effect of the protein on DNA after oxidative stress. In conclusion, our data may help to outline the different mechanisms by which the Dsup protein works in response to different insults.

Multivalent binding of the tardigrade Dsup protein to chromatin promotes yeast survival and longevity upon exposure to oxidative damage

“…While IR can directly induce SSBs and DSBs, much of its genotoxicity is mediated by hydroxyl radicals (OH • ), the most powerful oxidant among the reactive oxygen species (ROS) and generated when radiation interacts with water molecules 6 . Consistent with Dsup protecting 3 against hydroxyl radicals, it also reduces the number of DNA breaks in human cells exposed to hydrogen peroxide (H2O2) 4,7 . The high-energy hydroxyl radicals react with DNA bases to form lesions (including 8-oxoguanine; 8-oxo-G), while oxidation of the deoxyribose backbone dissociates sugar-phosphate bonds leading to DNA breaks 8 .…”

“…Indeed, Dsup binds with higher affinity to reconstituted chromatin over free DNA, and sequences within aa 360-445 are required for the association with chromatin and protection from DSBs caused by hydroxyl radicals 18 . While Dsup induction in human cells upregulates the expression of DNA repair genes 7 ; the protein also physically prevents DNA damage via chromatin binding, as this ability is observed in a reconstituted system lacking DNA repair factors 18 .…”

“…Tardigrade Dsup expression in human and plant cells alters transcription factor binding and gene expression in response to DNA damage 5,7 . This suggests that Dsup may bind preferentially to certain areas of the genome to influence gene expression.…”

“…~10 7 exponentially growing yeast cells (ODl600 0.8-1.0) were collected by centrifugation, washed once with water, and flash frozen in liquid nitrogen before being resuspended in 100 μL modified Laemmli buffer 51 and boiled for five minutes. Proteins were resolved by 10% SDS-PAGE, membrane transferred, and immunoblotted with antibodies to FLAG (Sigma F1804, 1:1,000) and GAPDH (Sigma A9521, 1:10,000).…”

Synergic homology directed recombination by PRDM9 meiotic factor

“…Expression of a dominant mutant variant of 53BP1 (DN1S) also inhibit NHEJ, by suppressing 53BP1, a key regulator of DSB repair pathway choice in eukaryotic cells and functions to favor NHEJ over HDR (Jayavaradhan et al 2019) (Canny et al 2018). Dsup is a Tardigrade protein that modulates DNA damage repair and was also included (Ricci et al 2021). DMC1 was also selected for the library, since this is a meiosis specific protein implicated in the repair of DSB using the homologous chromosome as the template for repairing the break (Hinch et al 2019)(Cole et al 2012).…”