Study Of PRRX1's Role In Regulating Non-homologous End Joining

Tumors threaten human health,and are the second leading cause of death.Genomic instability caused by DNA damage is one of the critical factors for tumor development and variation.The DNA double-strand break(DSB)is the most serious type of DNA damage,and non-homologous end joining(NHEJ)is the main repair method upon DSBs.In tumor cells,the NHEJ pathway is abnormally active to prevent tumor cells apoptosis,maintain tumor cells growth,and resist radio-and chemotherapies.Elucidating the specific molecular mechanisms of NHEJ repair in tumor cells can promote the development of new therapeutic methods to enhance the sensitivity of tumor cells to radio-and chemotherapies,improve treatment effects,and prolong patients lives.This study found that the transcription factor PRRX1 bound to the non-homologous end joining(NHEJ)core components Ku70,Ku80 and DNA-PKcs;and knockdown of Ku70 significantly down-regulated the protein level of PRRX1 in tumor cells,indicating that Ku70 may stabilize PRRX1.We used pulsed subcellular laser irradiator to damage living cells DNA,there were found that endogenous and exogenous PRRX1 were recruited to the damage sites within a few seconds and co-localized with Ku70,suggesting that PRRX1 may participate in the NHEJ pathway to repair DNA damage.In order to test whether the binding of PRRX1 to the damage sites is dependent on the recruitment of Ku or other proteins,we knocked down the expression of Ku70 and found that PRRX1 rapidly enriched to the DNA damage sites;after inhibiting PARP1 enzyme activity,PRRX1 was still able to rapidly enrich to DNA damage sites,these datas demonstrated that PRRX1 accumulated to DNA damage sites were independent on the recruitment of Ku and PARP1.PRRX1 is a member of the homeodomain(HD)family,and its three amino acids at positions 144-146 in the HD domain(Asn144,Arg145,Arg146,abbreviated as NRR)mediated the binding of PRRX1 to downstream target gene promoters,thereby affecting the transcriptional activation of PRRX1.We hypothesized that the NRR sequences may mediate the binding of PRRX1 to the DNA damage sites,then we constructed the vectors of single-point mutation and three-point co-mutation of the three amino acids(Alanine replacement).Using pulsed laser subcellular irradiator to damage living cell DNA,we found that single-point mutations of Asn144 and Arg146 and three-point co-mutation of NRR could significantly inhibit the degree and rate of PRRX1 enrichment to DNA damage sites,which demonstrated that Asn144 and Arg146 of PRRX1 were important sites to determine the accumulation of PRRX1 into the DNA damage sites.Further explored whether PRRX1 plays a crucial role in regulating NHEJ repair of DNA damage,we performed X-ray irradiation on tumor cells.As a positive control,we knocked down Ku70 in U87 MG cells,and found the phosphorylation level of serine 2056 of DNA-PKcs(DNA-PKcs-pS2056)was significantly down-regulated,which is a vital indicator of NHEJ activity,and PRRX1 was down-regulated too.Parallel knocking down of the expression of PRRX1 in U87 MG cells,we found that the expression of Ku70 was down-regulated after DNA damage;and the level of DNA-PKcs-pS2056 was also significantly down-regulated.These results suggest that PRRX1 may have a similar and redundant functions to Ku,and regulate each other after after DNA damage.In summary,PRRX1 regulated the NHEJ repair pathway by binding to the NHEJ core molecules Ku70/Ku80 and DNA-PKcs.This study enriches the understanding of DNA damage repair mechanisms and helps to develop new tumor treatment methods.