Preliminary Study On The Function Of SSART3 In DNA Damage Repair Pathway

The maintenance of the genome sequence is very important for the continuation of human life,and mutations also play a very important role in the maintenance and evolution of the genome,but at the same time,mutations can also lead to cancer,some human diseases and aging.As we all know,DNA is the basic unit of heredity.As an inherently reactive molecule,it is highly sensitive to endogenous and exogenous chemical modifications,so cells are potentially at risk of adverse mutations.To deal with these risks,cells are equipped with a complex array of pathways including DNA repair,injury tolerance,cell cycle checkpoints and cell death to reduce the adverse consequences of DNA damage.Cells respond to DNA damage by activating the powerful DNA damage response(DDR)pathway,which allows specific DNA repair pathways enough time to physically eliminate DNA damage in a substance-dependent manner.Therefore,further exploration of proteins involved in DNA damage repair and their mechanism of action is of guiding significance for the exploration of cancer and some human diseases.Recently,there is some evidence that SART3(Squamous Cell Carcinoma recognized By T cells 3)plays an important role in a variety of biological processes.Including but not limited to pre-mrna splicing,regulation of viral and host gene activation and transcription,regulation of protein degradation and regulation of cell survival,proliferation and differentiation.In addition,it has been reported that SART3 also plays an important role in trans-damaged DNA repair.In this study,we through the building with GFP plasmid of green fluorescent labels,using laser induced DNA damage living cells,under the multi-dimensional image acquisition software and analysis to explore the fluorescence dynamic changes in the cell nucleus,we can analyze according to the dynamic variation with GFP label SART3 protein can compare to quickly raise to sites of DNA injury.Then,we induced DNA damage in living cells by laser,and further confirmed the recruitment of SART3 at the DNA damage site by immunofluorescence assay.These results indicate that SART3 can recruit to DNA damage sites and may play an important role in DNA damage repair.By observing the dynamic recruitment of SART3 protein at the DNA damage site in the nucleus,it was not difficult to find that SART3 could be recruited to the damage site very quickly,and began to disperse in about 4 minutes until it recovered to the level before the damage.This suggests that SART3 may play an important role in DNA damage repair.We then constructed a series of mutants of SART3 with GFP fluorescence label in order to continue the study of the correlation between various domains of SART3 and recruitment at laser-induced DNA damage sites in living cells.The results showed that PEGFP-C1-flag-SART3 D2,PEGFP-C1-flag-SART3 D5 and PEGFP-C1-flag-SART3 D8 could not participate in the repair of DNA damage sites after laser-induced live cell injury.Since we have demonstrated through a series of experiments that SART3 is involved in DNA damage repair,but the exact mechanism of action is not clear,we used several common chemotherapeutic drugs to treat cells knocked down by SART3-Si RNA.To preliminarily determine SART3 was mainly involved in which chemotherapeutic drug induced DNA damage response.The results showed that the resistance of the experimental group to the chemotherapeutic drug cisplatin was significantly higher than that of the control group(wild-type cells).Subsequently,we found from protein immunoimprinting experiments that the protein level of SART3 in cells was significantly increased after cisplatin treatment,indicating that SART3 was associated with DNA damage repair induced by cisplatin.To further explore the role of SART3 in DNA damage repair,we analyzed the recruitment of SART3 at DNA damage sites in a mouse fibroblast model with DNA damage repair related gene defects.It was found that the recruitment of SART3 in PARP1 deficient cells was significantly reduced compared to that in the wild-type cells.After treatment with THE PARP inhibitor Olaparib,we found that the recruitment of SART3 at DNA damage sites was also significantly reduced.These results suggest that the involvement of SART3 in DNA damage repair may be regulated by the DNA damage repair factor PARP1.