Soutenance de thèse : Nuclear distribution and dynamics of the NTH1 DNA glycosylase before and after oxidative stress

Par Mrinalini Lianne Rao (IBS/Groupe Imagerie Intégrée de la Réponse au Stress)

In eukaryotic cells, nuclear DNA is constantly subjected to oxidative stress induced by reactive oxygen species (ROS), produced endogenously or by chemotherapeutic agents like cisplatin. To preserve genome stability, cells have evolved several DNA repair systems, notably the Base Excision Repair (BER) pathway, which eliminates small, non-helical distorting base lesions. BER is initiated by DNA glycosylases that recognize and excise the damaged bases, producing apurinic/apyrimidic sites, which are further processed to ultimately incorporate new and undamaged bases. Our team is particularly interested in NTH1, a bifunctional DNA glycosylase that recognizes and removes oxidized pyrimidines such as thymine glycols. Dysregulation of NTH1 expression or activity can lead to genomic instability and tumorigenesis. This thesis investigates the nuclear distribution and dynamics of NTH1 in cancer cells, before and after exposure to oxidative stress, using advanced fluorescence microscopy techniques, including confocal and single-molecule localization microscopy (SMLM), to achieve high spatial resolution.

For the first time, this study identified two distinct NTH1 populations : a freely diffusive nucleoplasmic pool and a chromatin-associated fraction forming foci. Using direct Stochastic Optical Reconstruction Microscopy (dSTORM, 10 nm resolution), we further characterized these two populations at nanoscale resolution, revealing the existence of dense chromatin-associated clusters of NTH1 as well as more sparsely distributed NTH1 molecules. The foci observed by confocal microscopy were found to be typically composed of at least two NTH1 clusters. Following oxidative stress, we observed a two phase response. First, immediately after exposure to a strong oxidizing agent, chromatin-associated levels of NTH1 as well as the total number of NTH1 foci and clusters per nucleus were found to decrease. Second, between 4 and 8h post-treatment, chromatin-associated levels of NTH1 along with the number and density of NTH1 foci and clusters increased significantly, suggesting a major re-organization of NTH1 in response to oxidative DNA damage. Single-particle tracking experiments further revealed two populations of NTH1 molecules : (i) a mobile, slow-diffusing population, likely transiently chromatin-bound, whose proportion and diffusion coefficient increased markedly after oxidative stress, before returning to control levels upon recovery and (ii) a largely immobile, likely tightly chromatin-bound, whose diffusion coefficient decreased 4h post treatment. Together, these findings support a change in the mode of diffusion of NTH1 in the nuclear space in response to oxidative DNA damage. Preliminary dual-labelling with chromatin markers and BER factors is underway to further explore foci composition.

Altogether, our work has contributed to a better understanding of the spatial and temporal distribution and dynamics of NTH1, providing insight into the tight regulation of NTH1, in response to oxidative stress.