Molecular Mechanism Of PTEN In DNA Damage Repair Regulation

Objective DNA damage repair is an important mechanism for maintaining the stability of the genome when cells are stimulated by DNA damage.Previous studies have shown that PTEN can promote DNA damage repair,but its molecular mechanism is not very clear.This study mainly uses different mutant forms of PTEN to explore its molecular mechanism in regulating DNA damage repair,including 1)the response of SUMO modification of PTEN to DNA damage repair;2)the function of phosphatase activity of PTEN during DNA damage repair.Methods 1)sh RNA and CRISPR-Cas9 techniques were used to construct PTEN knockdown or knockout cell lines,and then different forms of PTEN,including PTEN-WT,PTEN-C124S,PTEN-G129E,PTEN-K254R（SUMOylation site mutant）and PTEN-K266R（SUMOylation site mutant）,were re-expressed in those cells.Immunofluorescence staining（IF）was used to detect protein markers for DNA damage repair after Zeocin or irradiation treatment,includingγH2AX,53BP1 and RAD51.The DR-GFP and EJ5-GFP systems were used to detect the homologous recombination（HR）and non-homologous end joining（NHEJ）efficiencies;2)The Ni²⁺-pull down assay was used to detect the SUMO modification level of PTEN during DNA damage repair and the regulation of PTEN SUMOylation by p14^ARF.The RAD51 bound to chromatin was detected by chromatin separation;3)DNA damage-induced 53BP1 phosphorylation level was detected by Western Blot.In vitro dephosphorylation reaction assay was used to detect the dephosphorylation efficiency of 53BP1 by PTEN;4)Laser micro-irradiation was used to detect the recruitment of PTEN at the site of DNA damage.Co-immunoprecipitation（Co-IP）was used to detect the binding of PTEN with BRCA1 and p14^ARF;5)FACS was used to detect the apoptosis induced by Hydroxyurea（HU）,Camptothecin（CPT）and Zeocin in DU145 cells stably expressing different PTEN mutants.Results 1)Under DNA damage stimulation,γH2AX and 53BP1 foci as well as NHEJ efficiency were increased while RAD51 foci and HR efficiency were decreased in PTEN knockdown cells.The HR efficiency of PTEN-C124S,PTEN-K254R or PTEN-K266R re-expressing cells was lower than that of PTEN-WT and PTEN-G129E;2)Compared with PTEN-K254R and PTEN-K266R,SUMO modification of PTEN-WT,PTEN-C124S and PTEN-G129E were enhanced and modified bands obviously appeared on chromatin in response to stimulation of DNA damage.PTEN SUMOylation was promoted by p14^ARF via its interaction with PTEN,which was enhanced by Zeocin treatment;3)Under CPT,Zeocin,or irradiation treatment,PTEN knockdown and PTEN-C124S re-expressing cells had higher p T543-53BP1 levels compared to those of PTEN-WT and PTEN-G129E re-expressing cells.Cell-derived53BP1 p-T543 phosphorylation and in vitro synthesized 53BP1 phosphorylated polypeptide could be dephosphorylated by PTEN-WT and PTEN-G129E but not by PTEN-C124S;4)PTEN was recruited to the laser-induced DNA damage site.The binding of PTEN to the BRCT domain of BRCA1 was found and this interaction was enhanced by Zeocin treatment;5)HU and CPT treatment promoted apoptosis in PTEN-knockout,PTEN-C124S and PTEN-G129E re-expressing cells,and Zeocin treatment increased apoptosis of PTEN-knockout,PTEN-C124S,PTEN-K254R and PTEN-K266R re-expressing cells.Conclusion During DNA damage repair,PTEN SUMOylation mediated by p14ARF promoted the formation of RAD51 foci in the HR pathway;on the other hand,PTEN with its protein phosphatase activity could dephosphorylate 53BP1 to relieve the latter’s inhibitory effect on HR repair.Furthermore,stimulation of DNA damage significantly promoted the apoptosis of PTEN-deficient or mutated cells.Based on the above conclusions,this study could provide a theoretical basis for the clinical development of precise treatment strategy for tumor patients with PTEN deletion or mutation.