The Functional Research Of Protein Methylation Modification In DNA Damage Response

DNA damage response is an important signal transduction pathway in cells and an important factor in maintaining genome stability in cells.Protein methylation modification is a common protein post-translational modification,mainly including arginine methylation and lysine methylation,and plays an important role in DNA damage signaling.However,to date,there is still a lack of global profiling of whole-cell methylation changes during the DNA damage response and repair.In this study,using Hydrophilic Interaction Chromatography(HILIC)affinity enrichment combined with Mass Spectrometry(MS)analysis,we conducted a quantitative analysis of the methylated proteins in HEK293 T cells in response to Ionizing Radiation(IR)-induced DNA damage.In total,235 distinct methylation sites responding to IR treatment were identified,and 38%of them were previously unknown,including ADT2,MRNIP,PAPS1,BAG3,SPF27/BCAS2,RAD50,UBP7,FANCI,etc.In addition,through a series of bioinformatics analyses of the proteins identified by mass spectrometry,we found that RNA binding proteins(RBPs)were the most abundant proteins in the IR-induced methylome.Using the IPA database to conduct pathway analysis on the identified proteins,we found that the CSDE1 signaling pathway and the senescence pathway were the most prominent pathways up-regulated after IR treatment.Protein interaction(PPI)network analysis was performed through the STRING database,and it was found that the most abundant protein network was the m RNA processing pathway.Significant protein analysis of the identified proteins revealed that the proteins most significantly up-regulated in methylation in IR-treated cells included RING1,PARP1,PABP4,EEF1A2,XRN2,EF2,ADT2,and DHX9.Proteins with significantly down-regulated methylation include RA1L2/HNRNPA1L2,FBL,RBP56,SFPQ,THOC4/ALYREF,etc.These analyses provide a reference for subsequent studies on methylated proteins in response to DNA damage.In this study,we attempted to perform immunoprecipitation experiments on the DNA damage response protein PARP1 and identify its methylation sites by mass spectrometry.We obtained 10 different mono-methylation sites(K23,K59,K165,R173,K209,K254,K320,K418,K579,and K796).Among them,the level of K23 mono-methylation was approximately 4 times higher in IRtreated cells,and this site has been reported with no other PTMs yet.The mass spectrum of the K23 monomethylation site showed high confidence.Next,we verified the function of K23 monomethylation in DNA damage repair.This study validates the function of PARP1 K23 monomethylation in repairing IR-induced DNA damage.The results suggest that K23 A methylation deficiency sensitizes cancer cells to ionizing radiation and HU-induced DNA damage,and that PARP1 K23 methylation is involved in resolving replication fork block by regulating the binding of PARP1 to damaged forks.In conclusion,this study used mass spectrometry screening to identify methylated proteins in response to IR-induced DNA damage,and performed a series of bioinformatic analyses on the screened proteins to provide a reference for the study of methylated proteins in DNA damage response.On the other hand,this study revealed the role of PARP1 K23 methylation in the DNA repair response,and provided a foundation and basis for the subsequent resolution of the problem of resistance to PARP inhibitors.