Drosophila intestinal stem cell response to DNA damage and replication stress
Year: 2021
Type: dissertation
Abstract: Genome integrity in long-lived tissue stem cells is essential to maintain tissue function and prevent cancer initiation. How stem cells cope with DNA lesions determines their mutation rate, susceptibility to cancer, and likely age-related functional decline. My thesis aimed to understand what are the DNA damage causing factors, and what mechanisms are acting in adult stem cells to prevent spontaneous mutation. Replication stress driven by nucleotide depletion is proposed to increase genome instability in cancer cells, as it may lead to genome rearrangements. However, to what extent replication stress is responsible for genomic alterations in adult stem cells remains unclear. Likewise, the erroneous DNA repair mechanisms acting to repair DNA damage and linked to genome rearrangements are not completely understood. The Drosophila intestine is a good model system to study stem cells and tissue homeostasis and address these questions.The Bardin lab previously showed that different types of somatic mutations are arising in aging intestinal stem cells. Notably, wild type aged male guts frequently develop spontaneous neoplasias due to the inactivation of the tumor suppressor gene Notch by small or large deletions or more complex genomic rearrangements. During my thesis, I first examined how aged stem cells cope with DNA damage. I also helped to characterize the spontaneous formation of neoplasias in different genetic background. Microdissection and DNA extraction of neoplasias for subsequent whole-genome sequencing (work with K. Siudeja) and the development of bioinformatic pipelines (by N. Riddiford), allowed the characterization of the somatic mutations of intestinal stem cells. Since many structural variants identified suggested a DNA repair mechanism relying on microhomologies, I investigated the role of Pol θ, a polymerase involved in microhomology mediated DNA repair, in somatic mutations and neoplasia formation in intestinal stem cells.Several evidence from the sequencing suggested that replication stress might be an important cause of somatic mutation in the stem cells. Therefore, I examined the consequences of nucleotide depletion-induced replication stress on stem cells. Importantly, I developed a cell specific approach to induce replication stress by RnrL knockdown (an enzyme essential for the production of dNTPs). In the gut, the knockdown of RnrL induced DNA damage accumulation in S phase stem cells and proliferation defects, likely leading to stem cell loss. However, I observed that knockdown of RnrL in the developing wing disc rarely induced DNA damage cell autonomously. Following this, I demonstrated a role for GAP junctions in non-cell autonomously limiting replication stress, likely by buffering nucleotide levels between adjacent cells. Finally, I showed that GAP junctions are only localized between enterocytes in the midgut but not in stem cells. Differences in GAP junctions expression could explain tissue and cell specific sensitivity to replication stress and DNA damage.
Source: HAL
Author: Boumard, Benjamin
Language: English
Topic: DNA Repair Mechanisms
Subfield: Molecular Biology
Domain: Life Sciences
Open Access status: green
OpenAlex ID: W4392245983