The Mechanism Of P53^N236S Modulating Homologous Recombination Repair System

Genome instability is a“favorable feature”of cancer development.Compared with normal tissue cel s,the DNA damage and replication stress of precancerous lesions and tumor cel s are general y higher.As an important tumor suppressor gene,p53 not only regulates cell cycle arrest and induces apoptosis,but also modulates signal pathways such as DNA damage repair to maintain genome stability and inhibit tumorigenesis.However,about 1/2 of human malignant tumors have p53 deletion or mutation.And these mutations mainly occur in the DNA binding domain of p53 protein,resulting in p53 protein lost its sequence-specific transcriptional activity,leading to the ability of p53 to inhibit tumors is greatly reduced or even lost.Moreover,many p53mutations promote the occurrence and development of tumors and tumor drug resistance by gain of function（GOF）.In previous studies,we found p53^N236S(p53^N239S in humans,hereinafter referred to as p53S)in the fifth generation（G5）m Terc^-/-Wrn^-/-mouse embryonic fibroblasts（Mouse Embryonic Fibroblast,MEF）that can form telomerase-negative tumors.The mutation of p53S leads to the loss its signal pathway regulation function.Further research found that p53S mutant cel s were not sensitive to apoptosis that induced by doxorubicin（Doxrubicin,Dox,Topoisomerase??inhibitor）,suggesting that p53S enabled cel s to arise an anti-apoptotic phenotype.However,the research on the apoptosis pathway regulated by p53S suggests that p53S protein may not though apoptosis pathway result Dox induced p53S cell resistance.After further analysis of RNA-seq and Ch IP-on-Chip data,we found that p53S mainly activates two DNA damage repair pathways:homologous recombination（HR）and mismatch repair（MMR）.As a result,we focused on the damage repair mechanism that regulated by p53S In the DR-GFP homologous recombination system experiment,a higher HR level was detected in p53S cel s,which further provided clues for the p53S regulates the homologous recombination repair system.Therefore,this subject focuses on the regulation of p53S on genes related to HR repair system.In this study,we first detected that the expression level of HR key protein RAD51was higher in p53^S/Scel s than in wide-type and p53 knockout(p53^-/-)cel s by Western Blot.After low concentration Dox treatment,p53^S/S cel s mainly increased the expression of HR key protein RAD51 rather than the key protein 53BP1 of the NHEJ pathway to stress Dox-induced DNA damage.At the same time,immunofluorescence in situ staining result confirmed that p53^S/S cel s enhanced RAD51 signal after Dox treatment.The above results are consistent with RNA-seq and Ch IP-on-Chip expression profile data,suggesting that p53S can specifically transcriptional activate the expression of RAD51 protein.The results of cell cycle showed that p53^S/S cel s were mainly in G2 phase,and HR repair was mainly activatedin S/G2 phase.It suggested that p53S may promote the repair of Dox-induced DNA damage by enhancing the activity of HR.Next,we used the sh-m RAD51 plasmid to knock down RAD51expression in cel s.The RAD51 knockdown cell line wastreated by Dox,and the expression of apoptosis pathway-related protein Caspase 3,PARP,and DNA damage-related protein ATM,CHK2,andγH2AX were detected by Western blot,which showed that the DNA damage markerγH2AX increased significantly in p53^S/S cels after knocking down RAD51.Similarly,we detected a significant increase of apoptosis in Dox-treated p53^S/S cel s after knockdown of RAD51 expression by Annexin V-FITC/PI double staining flow cytometry.Subsequently,we used the immunofluorescence staining to observe that the signal ofγH2AX was increased in p53^S/S cels after knockdown of RAD51 and treated with Dox,and the colocalization of RAD51 andγH2AX in p53^S/S cels was significantly increased.It suggested that p53S up-regulates the recruitment of HR key protein RAD51 to damage sites to promote the repair of Dox-induced DNA damage and further develop drug resistance.We further detected the repair of damaged telomere in p53^S/S cel s.By transfecting HA-m TPP1^ΔRD plasmid into p53^-/-、p53^S/S MEF cel s to cause telomere damage.The result of TIF（Telomere-induced DDR foci）assayshowed fewer telomeres withγH2AX foci in p53^S/Scel s,suggesting a higher repair activity in p53^S/S,which reduced thetelomere damage,and this phenomenon was rescued in RAD51 knockdown cel s.At the same time,CO-FISH（Chromosome orientation fluorescence in situ hybridization）was used to observed the telomere sister chromatid exchange events（Telomere sister chromatid exchange events,T-SCEs）,and showed p53^S/S cel s with HA-m TPP1^ΔRDengaged higher T-SCEs,which decreased after knocking down RAD51,followed by increased telomere loss,suggesting that p53^S/S cel s can promote the repair of damaged telomeres by regulating RAD51-dependent HR.In summary,we preliminarily verified that p53S can regulate the expression of RAD51 and the recruitment at the DNA damage sites,thus enhancing the level of homologous recombination in cel s,and promoting DNA damage repair and telomere maintenance.The results of this study will help us to understand the drug resistance mechanism of p53S mutant tumors and provide new strategies for the target therapy of p53 mutant tumors.