Tudor-SN Promotes DNA Damage Repair Via Regulating Chromatin Remodeling

Objectives:Various stimuli in environment and endogenous stress in the cells can seriously affects the intracellular genetic information and gene coding methods.when suffering stimulation,the cells will activate a delicate process,including activation of DNA fragmentation recognition,checkpoints,DNA repair,chromatin remodeling,cell cycle arrest and apoptosis to maintain the stability of the genome.Tudor-SN(Tudor staphylococcal nuclease),a transcriptional co-activator,has many functions in cellular processes,such as pre-mRNA splicing,cell cycle regulation,stress granular formation,lipid metabolism,tumor invasion and migration.Recent studies have reported that Tudor-SN was related with apoptosis and chemotherapy resistance.Our previous results also showed that Tudor-SN can bind to some DNA damage repair protein.The main purpose of this project is to investigate the function of Tudor-SN in the early stage of DNA damage repair and detect the potential molecular mechanism.Methods: This project is divided into six parts.First,investigate the interaction between Tudor-SN and DNA damage repair proteins by using mass spectrometry and Co-IP in different cell lines treated with IR.In the second part,the relationship between Tudor-SN and cell survival after IR was detected by MTT assay,clone formation assay and apoptosis.In the third part,the recruitment and modification of Tudor-SN by PARP1 after injury was described by Co-IP,GST-pull down,also use Laser irradiation to detect the accumulation of Tudor-SN at DNA damage site.Then in the fourth part,the chromatin relaxation function of Tudor-SN in the nuclear injury site was explored by Co-IP and ChIP assay.In the fifth part,Tudor-SN function in activating the ATM-H2 AX and its downstream pathway was observed by western blot and con-focal microscopy.At last,we detected the Tudor-SN function in cell cycle arrest,HR and NHEJ repair pathways.Results: 1.Tudor-SN protein physically associates with a group of DNA repair protein.Mass spectrometry and Co-IP detection found that Tudor-SN binds to a variety of damage repair proteins(MDC1,RAD50,Ku70,53BP1,XRCC1,POLB).2.Tudor-SN depletion sensitizes cells to DNA damage and apoptosis.MTT and colony formation assays revealed that Tudor-SN deletion resulted in decreased survival rate of cells after IR,flow cytometry and western blot also showed the apoptosis increased in MEF-KO cell.3.PARP1 interacts with and poly(ADP-ribosyl)ates Tudor-SN in response to DNA damage,PARP1 can promote the Tudor-SN accumulation in damage site.4.Tudor-SN accelerates chromatin relaxation in response to DNA damage.Co-IP experiments showed that Tudor-SN could bind with SMARCA5 and GCN5 which can change the acetylation level of histone and then affect chromatin relaxation.5.The activation of ATM-H2 AX and its downstream signal pathway is remarkably inhibited due to un-sufficient chromatin relaxation impaired by loss-of-function of Tudor-SN protein.Con-focal results showed that Tudor-SN co-localized with γ-H2 AX in the nucleus after injury.6.DNA repair deficiency occurs in the cells with loss-of-function of Tudor-SN.Tudor-SN depletion results in abortion of G2/M arrest and impaired the DNA repair ability.In addition,loss of Tudor-SN leads to reduced GFP expression in DR-GFP-U2 OS cells and NHEJ-GFP-HEK293 cells after IR.Conclusion: 1.Tudor-SN is a DNA damage response(DDR)-related protein and plays important roles in the early stage of DDR.The loss-of-function of Tudor-SN exhibited cell survival deficiency and sensitive to IR.2.Tudor-SN can be recruitment to DNA damage sites in a poly(ADP-ribosyl)ation dependent manner via interaction with PARP-1.3.The accumulated Tudor-SN further recruited SMARCA5 and GCN5 to DNA damage sites,resulting in chromatin relaxation,and consequently activating the ATM and downstream DNA repair signaling pathways to promote cell survival.4.Tudor-SN can participate in both HR and NHEJ repair pathways to promote cell survival after IR injury.