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Abstract

Cells across all domains of life are persistently challenged by both endogenous and exogenous sources of DNA damage. Most eukaryotes rely on robust repair and tolerance mechanisms, yet these pathways are inherently limited and can be overwhelmed in high-stress environments. Extremotolerant organisms such as tardigrades have evolved additional protective factors. For instance, it has been reported that the tardigrade-specific DNA damage suppressor (Dsup) protein binds to nucleosomes and protects DNA against reactive oxygen species and ionizing radiation. This thesis investigates the function of tardigrade Dsup in the budding yeast Saccharomyces cerevisiae, a well-established eukaryotic model, to assess both its protective benefits and the cellular trade-offs that accompany its expression. In order to achieve stable expression of Dsup in yeast, I developed and optimized diverse strategies for introducing and expressing this protein into yeast. I tested centromeric and integrated constructs under both constitutive and inducible promoters. After optimizing expression, I studied Dsup’s impact on yeast physiology using a variety of phenotypic assays, as well as transcriptome profiling, chromatin mapping, and genome-wide genetic interaction screens. Although yeast cells expressing Dsup showed enhanced resistance to diverse oxidative and genotoxic stresses, there were also conditions where Dsup’s presence was not protective and could even be detrimental. At the cellular level, Dsup reprograms transcription, modifies chromatin accessibility, albeit slightly, and generates genetic dependencies in DNA repair pathways. Together, this work provides an integrated study of Dsup in a eukaryotic model using a wide range of phenotyping and multi-omics experiments. The combined results show that while Dsup retains protective functions outside its native context, these benefits are often counterbalanced by fitness trade-offs that emerge in the host cell. The findings emphasize both the promise and the limitations of expressing a heterologous nucleosome-binding protein. It offers new insight into the biology of the extremotolerance factor Dsup and its potential applications in genome protection and biotechnology.