Preliminary Study On The Function Of RCC2 In DNA Damage Repair

The cells in our bodies are under stress from DNA damage all the time in our daily lives.UV irradiation,ionizing radiation,DNA replication mismatch deletion and other factors can directly lead to DNA damage.After DNA damage,the genome will become unstable.If DNA damage cannot be repaired in time,the cell will have cycle arrest,and more seriously,it will lead to cell apoptosis.DNA damage repair is also closely related to the occurrence and development of cancer.Unstable genomes can cause normal cells to become cancerous.Therefore,DNA damage repair is of great significance in preventing cell cancerization.Up to now,more than 450 proteins have been reported to be involved in DNA damage repair.Further exploration and improvement of the mechanism of DNA damage repair have guiding significance for the treatment of cancer.RCC2(Regulator of chromosome Condensation 2)is a member of the RCC1 family of proteins,which contain one or more RLD(RCC1-like domain)structures,which play an important role in material transporting between nuclear and cytoplasm,protein ubiquitination regulation,cell cycle regulation and DNA damage repair,respectively.We screened RCC1 family proteins by constructing plasmids and expressing proteins with GFP green fluorescent tags in cells.We induced DNA damage in cells by laser irradiation,and the imaging results showed that RCC2 quickly recruited to the DNA damage sites.The recruitment of RCC2 at the DNA damage site was further confirmed by immunofluorescence assay using laser-induced DNA damage.This suggests that RCC2 may play a very important role in the repair of DNA damage.RCC2,also known as TD60(Telophase Disc-60),was originally identified as a telomere binding protein during mitosis,with a total length of 522 amino acids,containing a nuclear localization domain and an RLD domain.RCC2 is spatially sevenbladed propeller-like structure,and is expressed in both cytoplasm and nucleus.Previous studies have shown that RCC2 plays a role in cell cycle regulation,cancer genesis and tumor metastasis.These effects also indicate that RCC2 plays an important role in DNA damage repair.By analyzing the dynamic process of RCC2 recruiting DNA damage sites in living cells,we found that RCC2 can quickly respond to DNA damage,recruit DNA damage sites within 30S,and can continue to stay at the damage site.In order to explore how RCC2 is involved in DNA damage repair,different types of cancer chemotherapy drugs were first used to treat RCC2-knockdown cells,and it was found that RCC2-knockdown cells were the most sensitive to cisplatin.Western blot assay showed that the expression of RCC2 was significantly increased in cells treated with cisplatin.RCC2 has also been reported in previous studies to be associated with resistance to cisplatin therapy.Then we investigated the effect of RCC2 on cell cycle and found that G1 phase decreased and S phase increased in RCC2 knockdown cells.Then,we synchronized the cell cycle at the junction of G1 phase and S phase by the method of double thymidine block.After releasing the pressure of drug,we observed the speed of cell cycle progression.It was found that the cell cycle progression of RCC2 knockdown cells was significantly accelerated.This suggests that the absence of RCC2 may affect the regulation of cell cycle.Next,we designed a series of mutants D1?D6 with GFP tag for the structure of RCC2.Through dynamic fluorescence analysis,it was found that the recruitment ability of mutant D2 and D3 at the DNA damage site was decreased.We then analyzed the recruitment of RCC2 to the DNA damage site in the genedeficient mouse embryonic fibroblast model,and found that the recruitment of RCC2 at the DNA damage site could not be observed in the PARP1 deficient cells.RCC2 recruitment at DNA damage sites was also reduced in cells treated with PARP1 inhibitors.The results of endogenous immunoprecipitation showed that RCC2 interacted with PARP1.This suggests that PARP1 is a key factor in regulating the recruitment of RCC2 to DNA damage sites.