Nuclear PTEN Attenuates The Efficacy Of Non-homologous End Joining Through Interfering With Binding Of Ku70 At Double-strand Breaks

Background: The tumor suppressor phosphatase and tensin homologue(PTEN)is characterized as a phosphatase.Depending on this property,PTEN is capable of antagonizing the phosphatidylinositol 3–kinase–protein kinase B(PI3K-PKB)signaling pathway in the cytoplasm,thereby suppressing cell growth,proliferation and survival.In addition to its function in PI3 K pathway,this phosphatase also plays a vital role in other physiological process such as mitochondrial respiration.With the deepening of PTEN research,its function of maintaining genome stability attracts extensive attention.Genome stability is closely related to the integrity of DNA structure.However,reactive oxygen species,ionizing radiation and other toxic factors may damage DNA strands,causing DNA breaks.Among these breaks,DNA double-strand breaks are the most detrimental injury.The double-strand breaks repair is mediated by homologous recombination(HR)and non-homologous end joining(NHEJ).Homologous recombination occurs in the S or G2 phase of cell cycle and is an error-free repair process using undamaged sister chromatid DNA as a template.Nevertheless,non-homologous end joining is a template-independent pathway.Therefore,this repair process is error-prone with alterations of DNA sequences.At present,even though PTEN is deemed as an important regulator of DNA double-strand breaks repair,its underlying molecular mechanism in homologous recombination and non-homologousend joining pathway remains unclear.Objective: Herein,the specific function of nuclear PTEN in homologous recombination,non-homologous end joining and the underlying molecular mechanism were investigated in this study.The occurrence of various cancers is often accompanied by absence of PTEN function.Therefore,the investigation of PTEN in DNA double-strand breaks repair may reveal the unusual DNA repair pathway of cancers deficient of nuclear PTEN.Accordingly,our investigation may provide a novel idea and theoretical support for clinical diagnosis and treatment regimen to cancer patients deficient of PTEN.Methods: Site-directed mutants of PTEN were made using the overlap-extension PCR method.Single cell gel electrophoresis assay was used to test the repair capacity of cells when suffering from DNA double-strand breaks.Immunofluorescence was used to test the quantity of phosphorylated H2 AX foci in cells.Western blot assay was used to test the expression of PTEN and other proteins relevant to homologous recombination pave or non-homologous end joining pathway.Incompatible end joining assay based on p UC19 plasmid and in vitro plasmid-based non-homologous end joining efficiency assay were used to test the efficacy of non-homologous end joining.Electrophoretic mobility shift assay was used to test the binding of Ku70 complex at biotin-labeled DNA probe mimicking DNA double-strand breaks.Protein-chromatin crosslinking immunoprecipitation was used to test Ku70 binding ability at DNA double-strand breaks.MTT assay was used to test the sensibility of cells to various DNA damage agents.Results: BT549 cell which lacks intrinsic PTEN was transfected by PTEN wildtype(WT),PTEN phosphatase-negative mutant(C124S)or PTEN SUMOylation-deficient mutant(K254R)plasmids respectively.Single cell gel electrophoresis assay,immunofluorescence of phosphorylated H2 AX and western blot of phosphorylated H2 AX showed that PTEN promotes DNA double-strand breaks repair.In addition,this function of PTEN is dependent on its nuclear localization rather than phosphatase activity.Western blot used to test the level of Rad51 protein in cellsharboring PTEN or its mutants revealed nuclear PTEN increase the efficacy of homologous recombination pathway.Incompatible end joining assay based on p UC19 plasmid and in vitro plasmid-based non-homologous end joining efficiency assay proved that nuclear PTEN attenuates the efficacy of non-homologous end joining pathway.Further study showed that the level of Ku70,a critical protein in non-homologous end joining,remains unchanged in cells with PTEN variations.However,electrophoretic mobility shift assay manifested that Ku70 complex binding to biotin-labeled probe decreased 2-3 folds in cells with nuclear PTEN than cells deficient of nuclear PTEN.Protein-chromatin crosslinking immunoprecipitation confirmed that nuclear PTEN impairs Ku70 binding ability to DNA.PTEN was down-regulated in MDA-MB-231 cells through si RNA-mediated knockdown.Compared with PTEN-knockdown MDA-MB-231 cells,electrophoretic mobility shift assay proved that PTEN impaired Ku70 complex binding to DNA in MDA-MB-231 cells.MTT assay showed that lack of nuclear PTEN in cells confers cell hypersensitivity to DNA damage drugs.Conclusion: Our research elucidated a novel role of nuclear PTEN in maintenance of DNA integrity: PTEN is able to attenuate error-prone non-homologous end joining through impairing binding of Ku70 at double-strand breaks.Meanwhile,PTEN can promote homologous recombination by up-regulating the level of Rad51 protein.Of note,these functions are dependent on its nuclear localization rather than its phosphatase activity.