RNF8 Promotes DSB Repair By Regulating P53

DNA molecules are genetically important substances that store a sufficient amount of genetic information and play a central role in maintaining genomic stability and integrity.External factors and internal factors can cause DNA damage.If DNA damage is not properly repaired,they can be converted into permanent mutations that can significantly increase the risk of cancer or cause cell aging or death.In order to cope with DNA damage,eukaryotic cells have evolved a series of response mechanisms,DNA-damage response(DDR),which allow DNA damage repair factors to be recruited in order to quickly identify damaged sites,thereby coordinating different cellular processes.Such as DNA repair,cell senescence and apoptosis.DNA-double-strand breaks(DSBs)are the most serious form of DNA damage,and exogenous factors can occur in normal physiological processes.Unrepaired DSBs have dangerous consequences,such as inducing genomic instability and promoting apoptosis or tumorigenesis.The two classical pathways for repairing DSBs are Homologous re-combination(HR)repair and nohomologous end-joining(NHEJ)repair.RNF8 is an important protein for DNA damage response and an E3 ubiquitin ligase.RNF8 plays a central regulatory role in the upstream of the HR and NHEJ repair pathways,and promotes DNA damage repair by recruiting downstream damage repair proteins by ubiquitination of histones.p53 is an important protein that maintains the stability of the genome and is one of the important anti-cancer genes in the human body.Under normal conditions,the intracellular p53 content is low.When DNA damage occurs,p53 is activated,enters the nucleus,participates in the regulation of transcription of related genes and participates in downstream events such as growth arrest,DNA repair,apoptosis,and senescence.RNF8 and p53 play an important role in DNA damage repair,so what is the relationship between them in the process of DSB damage repair?In this study,we used Etoposide(ETO)as a DNA-damaging drug to induce DNA double-strand break damage in cells,and studied the effect of RNF8 on p53 function.We found that the cell survival rate of RNF8 was decreased,and the apoptosis rate was increased,which means that the deletion of RNF8 impaired DNA damage repair.However,low expression of RNF8 promoted an increase in the protein level of p53.The low cell survival rate of RNF8 and p53 was decreased,and the apoptosis phenomenon was decreased.This indicates that the increased sensitivity of EOF to cells with low expression of RNF8 may be caused by up-regulation of p53 protein.Next,we used DR-GFP-U2 OS and EJ5-GFP-U2 OS two reporting systems to detect the effects of RNF8 and p53 on HR and NHEJ repair efficiency of DSB injury by flow cytometry.The results show that RNF8 can promote DSB repair by regulating the function of p53.RNF8 promotes efficient DNA damage repair,and sustained activation of p53 promotes cell apoptosis.To investigate how RNF8 regulates the apoptosis-inducing function of p53,we performed mass spectrometry and co-immunoprecipitation and GST-Pull Down experiments,and found that RNF8 and p53 were present in the same complex.Western Blot and Real-time PCR experiments showed that RNF8 as E3 ubiquitin ligase did not mediate ubiquitination of p53,but regulated the level of p53 m RNA.Dual luciferase reporter gene experiments revealed that normal expression of RNF8 inhibits p53 transcription and promotes DSB damage repair.Taken together,we believe that RNF8 may regulate the apoptosis-inducing function of p53 by inhibiting the expression level of p53.This study preliminarily explored another pathway by which RNF8 promotes DSB repair.RNF8 may promote DSB repair by inhibiting the apoptosis-inducing function of p53,which lays a foundation for further revealing the role of RNF8 and p53 in DSB repair and the mechanism of interaction regulation.