TOWARDS THE DISCOVERY OF NOVEL PROTEINS AT THE APEX OF THE DNA DAMAGE RESPONSE

Abstract

Exploring the intricate pathways of DNA Damage Response (DDR) is crucial for understanding cancer formation and developing treatment strategies. DNA damage response and repair occur in the context of chromatin; therefore, it is crucial to study this process within the physiological context of an organism. Previous proteomics-based studies aimed to identify proteins involved in the early stages of DDR often utilize in vitro subcellular fractionation in specific cell lines. However, studying such processes outside the organism context overlooks the complexity and interactions of the physiological environment, potentially limiting the translatability of findings. Furthermore, protein abundance within each cellular compartment can obscure the detection of the less prevalent proteins, including those involved at the apex of the DNA damage response. To address these challenges and in search for the upstream novel regulators of DNA damage response in vivo, we have developed a methodology to isolate the native chromatin in its native conformation from the heterogeneous cell population of C. elegans. We have successfully isolated chromatin and chromatin-bound proteins by performing buffer-based fractionation of various cellular components, aiming to isolate the intact chromatin. Separation and purity of subcellular compartments were validated by electrophoresis analysis and Western blot using subcellular specific markers. Using SILAC-based quantitative proteomics, we have identified new proteins associated with DDR, which likely play a crucial role in recognizing and repairing damaged DNA. Additionally, by integrating our approach with affinity purification against labeled DNA breaks and proteomics, we explored the chromatin-bound proteins directly at the sites of DNA damage. This approach enabled us to identify several RNA helicases with potential role in DNA damage response. We strongly believe that this strategy can facilitate the exploration of the protein landscape at DNA damage sites and enhance our understanding of protein dynamics during DNA damage repair.

Date of Award	2026
Original language	American English
Awarding Institution	HBKU College of Health & Life Sciences