Identification of Novel Proteins Involved in the DNA Damage Response

The human genome is constantly exposed to exogenous and endogenous agents that can cause DNA damage and alter its integrity. It is essential to maintain genomic integrity to ensure proper cell function and faithful transmittance of genetic information. Therefore, cells counteract the effects of DNA damage by activating DNA damage response, a coordinated series of complex mechanisms including sensing, transducing, and activating the appropriate DNA repair pathway. In this study, we aimed to identify novel proteins that contribute to maintaining genomic stability through two independent experimental approaches: RNA interference (RNAi) reverses genetic screening and Stable Isotope Labeling by Amino acids in Cell culture (SILAC)-based mass spectrometry (MS) in Caenorhabditis elegans (C. elegans). Through reverse genetic screening, we screened 252 gene clones from the chromatin ontology subset of the Ahringer RNAi library. We identified and confirmed 25 genes that showed increased hypersensitivity to Hydroxyurea (HU). The identified candidates primarily function in chromatin silencing, DNA repair, and regulation of transcription, suggesting a potential involvement in DNA damage response signaling. Further, SILAC-MS analysis of C. elegans’ response to cisplatin revealed an upregulation of proteins associated with stress and detoxification processes, including Glutathione S-Transferase (GST) and complement C1r/C1s, Uegf, Bmp1 (CUB) domain-containing proteins. Overexpression of GST was previously reported to be associated with acquired resistance to cisplatin. Therefore, our data can provide a list of predictive biomarkers and drug targets for more effective and personalized patient treatments. In addition, the dataset we provided is considered a valuable source of information for the scientific community's future studies in DNA damage and drug response.