The Studies On The Function And Mechanism Of RNF8 And CtIP In DNA Double Strand Break(DSB) Damage Response

The structural integrity and functional stability of genomic DNA are important for the normal life activities of organisms.Eukaryotic genome will face external and internal adverse factors at any time,resulting in DNA damage.DNA double strand breakage(DSB)is one of the most serious types of damage,with homologous recombination(HR)and non-homologous terminal junction(NHEJ)repair pathways.In order to maintain the stability of genome,life has formed a set of perfect defense mechanism in the long-term evolution process,called DNA damage response(DDR).DDR activates and initiates a series of downstream effect events through a cascade signal amplification process,which work together to complete DNA repair,among them,DNA damage repair and cell cycle checkpoint activation are important events in the process of DSB damage response,but the specific functional mechanism still needs to be further explained.Many proteins are involved in DSB damage repair,among which E3 ubiquitin ligase RNF8 plays an important role.However,the specific functional mechanism of RNF8 is still unclear.We established a DSB damage model by means of etoposide(Eto)and restriction enzyme,etc.Through cell proliferation,apoptosis,comet electrophoresis and other experiments,it was found that low RNF8 expression increased cell sensitivity to DSB injury,and the increase of sensitivity to DSB damage was correlated with the increase of p53.Through DR-GFP and EJ5-GFP damage repair reporting system and mass spectrometry,it was found that RNF8 could improve the efficiency of DSB damage repair by inhibiting the pro-apoptosis function of p53,and the regulation of p53 apoptosis induction function was achieved by inhibiting the acetylation of p53-k120 site by Tip60.Once DSB injury occurs,cell cycle checkpoint will be activated immediately and lead to cell cycle arrest,providing enough time for the orderly repair of DSB damage.The abnormality of this process will not only affect the efficiency of DSB repair,but also affect the sensitivity of tumor to chemotherapy drugs in the process of tumor treatment.Endonuclease CtIP not only plays an important role in DSB damage repair,but also is a key cell cycle checkpoint regulatory protein,however,the specific mechanism of its regulation of cell cycle checkpoint is still unclear.Through flow cytometry,cell proliferation and Real-time quantitative PCR assays,we found that low CtIP expression in HCT116 cells decreased Eto induced G2/M cell cycle arrest and decreased sensitivity to Eto.The regulation of G2/M arrest of HCT116 cells by CtIP mainly depends on the ATR-Chk1-CDC25 C pathway,and is unrelated to the p53-p21/GADD45 a pathway.So far,we have discovered a new mechanism for the DDR response process.The DSB damage response is a series of process that activating of damage repair signals and cell cycle checkpoint.Once DSB damage occurs,the DDR process is activated,on the one hand,RNF8 can improve the efficiency of DSB damage repair by inhibiting the pro-apoptosis function of p53.On the other hand,CtIP can activate cell cycle checkpoint through the ATR-Chk1-CDC25 C pathway,leading to G2/M cell cycle arrest,providing sufficient time for DSB damage repair.RNF8 and CtIP mediate DNA damage repair and cell cycle checkpoint regulation together,to coordinate and eventually complete the repair of damaged DNA.This study deepened the understanding of DSB damage response process and strengthened the understanding of DNA damage repair mechanism,cell cycle checkpoint regulation mechanism and their mutual relationship.