The Function Of PARP1 And PTEN In DNA Damage Repair Pathway-NHEJ

PARylation as a universal proteins post-translational modification controls a wide array of cellular processes.PAPR1,the first founding member of the PARP super family,contains multiple functional domains and participates in the pathway of DNA damage repair.PARP1 as a therapeutic target,is applied to therapy of ovarian and breast cancer.It is still uncertain that the exact molecular mechanism of PARP1 regulating DNA damage repair and it has always been controversial that the role of PARP1 in DNA double strand breaks(DSBs)repair.Therefore,in our study we investigated that the function of PARP1 in controlling the DSB repair pathway-nonhomologous end joining(NHEJ).We found that the effect of PARP1 inhibitors were limited on the proliferation of cancer cells with wild-type BRCA gene.This was consistent with the strategy of clinical treatment of PARP inhibitors.In order to achieve same effect for the cancer cells with wild-type BRCA gene,we consider to inhibit the efficiency of homologous recombination(HR).RAD51 was an important protein in HR repair pathway.The inhibition of RAD51 would impair the efficiency of HR repair pathway.Hence,we combined the PARP inhibitor and RAD51 inhibitor to treat cancer cells.This strategy markedly improved the anti-tumor effect of PARP inhibitor and it could be a potential method of clinical treatment of cancer.Through overexpression of PARP1 in 293 T cell line we found that the efficiency of total-NHEJ pathway was upregulated and when separately overexpressing diverse domain of PARP1 in 293 T cells we confirmed that the promoting of total-NHEJ efficiency mainly depended on alternative-NHEJ pathway.The deficiency of functional domain of PARP1 would result in decreasing efficiency of NHEJ pathway.So,our study indicated that PARP1 promoted NHEJ pathway depending on its structure.In addition,through researching the structure of PARP1 showed that cleaved-PARP1 including P24 and P89 protein lost the function of recruiting in nuclei.This result show that cleaved-PARP1 impaired the nuclear location sequence(NSL)and was excluded from nuclei.Then we investigated the effect of PARP1 on Ku70 protein,a fatal protein in Classical-NHEJ pathway.Our results revealed that PARP1 could combine Ku70 in a physical method rather than depending on the conjugation of DNA.Besides,we found that PARP1 modified Ku70 depending on its enzymatic activity and this modification was regulated by PTEN.The PARylation of PARP1 was limited by wild-type PTEN and it resulted in compromising activation of PARP1,finally impairing the PARylation of Ku70 When the cells were treated with irradiation,the level of PARP1 decreased firstly and then increased with the time and recovered the normal level at 24 hours after exposing to IR in WT-PTEN cells.We found that the level of PARP1 was unaltered in PTEN deficient cells.However,the level of PARP1 was stable when we treated cells with a PARP inhibitor-olaparib in WT-PTEN cells.These results further illustrated that the variation of PARP1 depended on its enzymatic activity that modify PARP1 itself by PARylation.We detected the m RNA of PARP1 and there was no difference between PTEN-WT and PTEN deficiency cell lines.It demonstrated that PARP1 modified itself and activated PARP1 decreased the stability,then PARP1 was degraded by proteasome.So the level of PARP1 was down-regulated in PTEN-WT cells.To sum up,our study found PARP1 was involved in regulating NHEJ repair pathways and promoted the efficiency of A-NHEJ repair pathways by modifying PARylation of Ku70 protein and reducing the ability of combination with chromatin.We also found that the modification effect of PARP1 was regulated by PTEN.Our experimental results further illustrated the dual regulation mechanism of PAPR1 in NHEJ repair pathways and explained current problems about PARP1 proteins involved in DNA repair and provided a better reference of using PARP inhibitors for tumor cells with different genotypes.