The Study Of Ring Finger Protein 8 On The Choice Tendency Of DNA Double Strand Break Repair Pathway

DNA is an important genetic material in living organisms that controls many biological life activities such as genetics and metabolism.However,DNA is constantly attacked and destroyed by external and internal factors such as various environments and its own factors,resulting in various forms of damage.After DNA damage,the cells immediately initiate a set of precise response systems to deal with these injuries,preventing further negative effects on cell function and physiological activities.This is DNA damage repair.DNA damage repair is the physiological repair mechanism of damaged DNA by cells.DNA double-strand breaks(DSBs)are the most severe form of damage in various forms of DNA damage.There are two classic repair pathway to deal with DSBs damage,namely homologous recombination(HR)repair and nonhomologous end-joining(NHEJ)repair.If the broken DNA is not repaired in time,it will cause a series of physiological events to change,and the most serious situation will lead to functional defects,cancer,and death of the cells.RNF8(Ring finger protein 8)is an E3 ubiquitin ligase that plays an important role in DNA double-strand break injury repair.The recruitment of BRCA1 and 53BP1 is severely affected by interference with RNF8 expression,and the process of DNA double strand break injury repair is inhibited.Since RNF8 affects the recruitment of 53BP1 and BRCA1,and these two molecules determine the two options of NHEJ repair and HR repair,then whether RNF8 participates in these two pathways and how the two pathways affect choices.Does it have a tendency?In this study,we used a topoisomerase inhibitor ETO as an experimental drug to construct a DNA damage repair model.We found that 10 μM ETO stimulated U2 OS cells for 20 min,and the repairing effect was best when repaired for 1 h.After interference with RNF8 expression,the proliferation of cells was significantly inhibited,and the phenomenon of cell apoptosis was enhanced.This means that the deletion of RNF8 inhibited the damage repair of DNA and caused the abnormal physiological function of cells,resulting in increased apoptosis of physiological functions of cells,reduced proliferation.Subsequently,we detected the recruitment of RNF8 on chromatin and found that RNF8 recruitment on chromatin gradually increased after DNA damage.With the completion of DNA damage repair process,RNF8 gradually separated from chromatin.The results show that RNF8 participates in DNA damage repair process.After interfering with RNF8 expression,the recruitment of the Maker proteins BRCA1 and 53BP1 to the DNA damage repair pathway was also weakened,indicating that RNF8 is involved in the above two repair pathways and is in the upstream of pathway choice.To further explore the RNF8’s propensity to select two repair pathways,we used the laboratory’s reporter cells,DR-GFP-U2 OS and EJ5-GFP-U2 OS,to detect DNA damage repair pathways that RNF8 was involved in,The tendency of RNF8 to influence the two pathways was also examined.The DR-U2 OS system was used to detect homologous recombination repairs,and the EJ5-U2 OS system was used to detect nonhomologous end-joint repairs.The results show that RNF8 has a greater impact on the NHEJ pathway in the DNA damage repair pathway.The above results show that RNF8 is involved in DNA double-strand break repair and has a greater impact on the NHEJ pathway of DNA double-strand break repair.This study will lay the foundation for the study of RNF8’s selection of DSB damage repair pathways.