Tardigrades are extremophiles that withstand harsh environments through unique molecular strategies. One such strategy involves Damage Suppressor (Dsup), a protein shown to protect cells from radiation-induced DNA damage. Little is known about the biochemical and structural characteristics of Dsup that lead to DNA protection. To gain insight into the mechanism of DNA protection by Dsup, we examined its fundamental biochemical and structural properties using mass photometry, biolayer interferometry, small-angle X-ray scattering, and microfluidic modulation spectroscopy. We found that Dsup is largely intrinsically disordered and binds DNA with high affinity via a multi-valent interface. This interaction induced conformational changes in both Dsup and the DNA, suggesting a potential structural mechanism of its DNA protection ability. We propose that Dsup alters DNA structure, possibly by partially unwinding it, to reduce its susceptibility to damage. These findings offer new insights into how a disordered protein such as Dsup functions as radioprotectants in extreme environments.

Tardigrades are microscopic invertebrates capable of surviving extreme environmental conditions through unique molecular adaptations. Among the proteins implicated in their remarkable resilience is a novel protein known as damage suppressor (Dsup), a key factor in protecting cellular DNA from elevated levels of radiation. Since its discovery, numerous studies have explored the biochemical, structural, and functional properties of Dsup. In this review, we summarize the current knowledge surrounding these properties and describe several proposed mechanisms by which Dsup may confer protection. For each proposed mechanism, we outline the foundational model, present supporting evidence, and highlight critical gaps in our understanding. Taken together, we believe that Dsup likely employs multiple complementary mechanisms to protect DNA. Finally, we discuss emerging applications of Dsup and Dsup-inspired technologies for human health. Overall, this review synthesizes our current understanding and provides a framework to guide future investigations into this remarkable protein.

Translesion synthesis is a major DNA damage bypass pathway in which specialized DNA polymerases, such as pol η and Rev1, are recruited to stalled replication forks where they catalyze nucleotide incorporation opposite DNA damage. Translesion synthesis is regulated by the Rad6-Rad18 complex, which catalyzes PCNA mono-ubiquitylation. Despite its central role in regulating translesion synthesis, its oligomeric state and molecular interactions are poorly understood. Here, we use mass photometry to show that Rad18 co-purifies with Rad6 and forms a series of larger oligomeric complexes. Using yeast two-hybrid studies, we showed that while the full-length Rad18 complex interacts with pol η, its interaction with Rev1 is controlled by auto-inhibition. The release of this auto-inhibition is associated with the dissociation of the Rad6-Rad18 complex, freeing Rad18 monomers and dimers. Because pol η participates in non-mutagenic translesion synthesis, while Rev1 participates in mutagenic translesion synthesis, this auto-inhibition likely allows pol η preferential access to stalled replication forks by delaying Rev1 recruitment. Such an ordered polymerase-recruitment mechanism would reduce the likelihood of mutations.