Role And Mechanism Of CtIP In DNA Double Strand Break Damage Response

DNA is the genetic material in cells,and the structural integrity of DNA is essential for the normal function of cells.However,cells in an organism are often stimulated by endogenous or exogenous factors to cause DNA damage.Among the many types of DNA damage,DNA double-strand break(DSB)damage is the most serious type of damage.When DNA double-strand break damage occurs,the cells responds to the stimulations,which is called DNA damage response.Among them,a key response is cell cycle arrest.That is,DNA damage activates the cell cycle checkpoint and prevents the cell cycle from continuing.Cell cycle arrest provides efficient time for DNA repair,through which to prevent the cells with damaged DNA from entering the dividing phase.CtIP is a Ct BP interacting protein that was originally discovered as a transcription cofactor.It was found that CtIP is involved in the processes of DNA damage response,transcription regulation and tumorigenesis.At present,the research on the role of CtIP in the response to DNA double-strand break damage is mainly focused on DNA damage repair.CtIP has endonuclease function and transcription regulation function,and CtIP function cannot be separated from its own post-translational modification.Among them,the phosphorylation modification of checkpoint protein kinase ATM and cyclin kinase CDK is essential for the activity of CtIP.Studies have found that CtIP promotes cell cycle checkpoint signal transmission through the protein kinase Chk1.But the research on the function of CtIP in DSB-induced cell cycle arrest is still far from enough.In this study,Etoposide(ETO)was used as a DNA double-strand break damage drug,and experimental techniques such as Western Blot,Real time-PCR,cell cycle measurement,apoptosis measurement,and cell survival measurement were used.The role and mechanism of CtIP in cell cycle arrest in the DSB condition were explored.The main results are as follows:(1)We determined that CtIP participates in the response to DNA double-strand break damage by examining the effect of DNA double-strand break damage on CtIP expression,and the survival rate and apoptosis of DSB cells after knocking down CtIP.(2)The effect of CtIP on cell cycle checkpoints and the expression of cell cycle-related proteins in DSB were detected by knocking down CtIP,and it was found that CtIP promote G2/M phase arrest in DSB.(3)Through Real time-PCR and Western Blot experiments,it was found that CtIP affects the expression of p21,but does not affect the expression of p53,an upstream protein of p21.By measuring the G2/M phase arrest of cells in the absence of p53 single deletion and co-deletion of p53 and Ct IP,it was determined that CtIP does not promote cell G2/M phase arrest through the p53-p21 pathway in DSB.(4)We further found that the Ser327 mutation of CtIP weakens the G2/M phase arrest of the cells when DSB,and the absence of CtIP weakens the ATR-Chk1-CDC25 C checkpoint pathway,suggesting that the endonuclease function of CtIP is very important for the G2/M phase arrest caused by DSB.In this paper,Ct IP is involved in cell cycle arrest under DNA double-strand break damage and the related mechanism is explored.The results of this paper has provided new clues for the study of the role of CtIP in response to DNA double-strand break damage and added new information to basic research on cell cycle arrest.