Involvement of Rad51 paralogs in Rad51 filament formation in DNA repair
Homologous recombination (HR) is a major repair mechanism for DNA double-strand breaks induced by ionizing radiation. A key step in HR is the formation of Rad51 nucleoprotein filaments on the single-stranded DNA generated from these breaks. We have shown that strict control of these filaments is essential, so that HR does not itself induce chromosomal rearrangements (eLife 2018, Cells 2021). In humans, functional homologs of control proteins are tumor suppressors. Thus, the control of HR appears to be as important as the HR mechanism itself. Our project involves the use of new molecular tools enabling a real breakthrough in the study of these controls. We will be using a functional fluorescent version of the Rad51 protein developed for the first time by our collaborators A. Taddei (Institut Curie), R. Guérois and F. Ochsenbein (I2BC, Joliot, CEA). This major advance will enable us to observe the influence of control proteins on DNA repair by microscopy in living cells. We have also developed highly accurate structural models of control protein megacomplexes in association with Rad51 filaments. This study also led to the identification of specific domains for each paralog protein, outside the structurally conserved Rad51-like core, that might define the specificity of each paralog proteins. We will use a multidisciplinary approach based on genetic, molecular biology, biochemistry, protein structure and live microscopy methods and yeast as model organism to study the consequences of the ablation of these specific domains. We will also search for proteins specifically binding these domains. Their identification would be crucial to understand the function of Rad51 paralog complexes and help to develop new therapeutic approaches.
Localization and dynamics of key nucleoid-associated proteins during stress-induced bacterial nucleoid remodeling.
Nucleoid remodeling, and in particular, nucleoid compaction, is a common stress response mechanism in bacteria that allows bacteria to rapidly respond to sudden changes in their environment. Using advanced optical microscopy approaches, we recently followed the changes in nucleoid shape and volume induced by exposure to intense UV-C light in the radiation resistant bacterium, Deinococcus radiodurans. This two-step process involves a rapid initial nucleoid condensation step followed by a slower decompaction phase to restore normal nucleoid morphology, before cell growth and division can resume. Nucleoid associated proteins (NAP) are known to be key players in this process, although the details of their implication remain largely elusive. We have started to shed light on the central role of the major NAP, the histone-like HU protein, in this process. The proposed PhD project will extend this work to the study of 5 additional NAPs involved in stress-induced nucleoid remodeling. The PhD student will perform biochemical studies to follow the abundance of these key factors, live cell imaging to map their distribution and single-particle tracking to determine their dynamics. This work will contribute to a better understanding of the fundamental processes that govern bacterial genome organisation and how they are affected by UV radiation and DNA damage.
Effects of ionizing radiation and radiosensitizing molecules in a relevant murine model of breast cancer
The project aims at evaluating the efficacy of molecules combined with radiotherapy, in in vitro and in vivo models of breast cancer.
On the one hand, the student will evaluate the radioenhancer effect of bimetallic nanoparticles designed in the laboratory, on a murine model mainly. A clinical, histological, and immune monitoring will confirm the added value of such molecules for combination with radiotherapy. In addition, those innovative nanoparticles have been designed as biodosimeters, using unique physical properties of metallic nanoparticles. Therefore the project includes an evalution of the biodosimetry potential, in collaboration with physicists from CEA, who developed detection tools.
On the other hand, specific inhibitors for DNA repair will be used to block radiation-triggered repair. Thus, damaged cancer cells will be oriented towrads cell death, even in the case of radioresistant cells. The objective of the PhD program is to evaluate these molecules effect on in vitro cellular models, as well as on murine models of breast cancer.
Overall, the research project will benefit from the laboratorys’ collaborations with physicists and chemists, as well as the platforms of IRCM (irradiation, animal experimentation, microscopy, cytometry, etc...)
Impact of LET on biological response to Flash irradiations
Recent studies with electron and proton beams have shown that irradiation at dose rates above 40 Gy/s can be as effective in inhibiting tumor growth as irradiation at the conventional dose currently used (typically 1 Gy/min) but much less toxic to healthy tissues. This phenomenon is known as the “FLASH effect”. This effect is considered one of the most important discoveries in the recent history of radiobiology due to its potential to improve the therapeutic window between tumor control and normal tissue toxicity. Recent studies show that the biological mechanisms of the FLASH effect are linked to differential tissue oxygenation. However, the exact mechanisms of the cellular biological effects of FLASH irradiations are not completely clear and some are even contradictory.
The objective of this project is a molecular characterization of the FLASH effect on a model system perfectly controlled in vitro. FLASH irradiations of cancer cells and healthy cells will be compared to conventional dose rate irradiations using electrons and carbon ions in the two associated laboratories. The differential effect will be related to the oxygenation condition of the cells, REDOX/mitochondrial metabolism and general changes in cellular metabolism.
Response of Spermatogonial Stem Cells to heavy ion irradiation: functional assessment and transcriptome profiling in adult mice.
In deep space, astronauts will be exposed to galactic cosmic rays, whose high-energy heavy ions - minority elements - are highly toxic to cells. The consequences for the body of this chronic, low-dose exposure are still poorly understood, due to a lack of human data. In order to assess the impact of a prolonged stay in space on male fertility, this project proposes to study the effects of irradiation by a 56Fe ion beam on mouse spermatogonial stem cells (SSCs). In adults, the continuous production of spermatozoa relies on a stock of SSCs maintained by self-renewal. The integrity of the activity of irradiated CSS will be tested in vivo using transplantation tests. Various parameters of irradiated CSS will be analysed (DNA lesions, mortality, cell cycle, etc.). A transcriptional signature and markers of exposure to heavy ions will be sought in undifferentiated spermatogonia (single cell - RNA seq), and the gene networks involved in stress responses will be studied in particular. All of this data could serve as a basis for studying the hereditary and epigenetic risk associated with space flights, as well as for improving protective measures.