APE1 Promotes DNA-PKcs-Mediated HnRNPA1 Phosphorylation And Its Role In Mitotic Telomere Protection

BackgroundMaintenance of the structural integrity of telomere is necessary to prevent the activation of the DNA damage response(DDR) and improper chromosome end-to-end fusion events, which in turn will impair chromosome segregation and cause aneuploidy. One of critical issues of telomere maintenance has been the transition between DNA replication and reestablishment of the capping by shelterin, particular at the single-stranded 3’ overhangs. Replication protein A(RPA) complex is the predominant single-stranded DNA binding protein and is essential for both DNA replication and damage repair. Upon stalled replication forks, extension of single-stranded DNA and the coating of RPA trigger the activation of ataxia-telangiectasia and Rad3-related(ATR) kinase and DDR. Thus, it is critical to displace RPA from the newly replicated telomeric 3’ overhangs to prevent unnecessary activation of the ATR signaling pathway at telomeres. Protection of telomeres 1(POT1), one of the shelterin components, binds to the single-stranded telomeric 3’ overhang and is required for suppression of ATR-dependent DDR. However, POT1 alone cannot out compete with RPA for the binding of single-stranded telomeric DNA and requires additional support from heterogeneous nuclear ribonucleoprotein A1(hn RNPA1) for the RPA-to-POT1 switch at single-stranded telomeric DNA.The involvement of hn RNPA1 in telomere protection has also been link to DNAdependent protein kinase(DNA-PK) since hn RNPA1 interacts with and could be the direct substrate of DNA-PK. On the other hand, DNA-PKcs is necessary to prevent telomere fusions but not the maintenance of telomere length, suggesting that DNA-PKcs is needed for the synthesis of telomere cap structure. Furthermore, DNA-PK is composed of the DNA-binding Ku70/Ku80 subunit and the catalytic DNA-PKcs subunit, and is the critical regulator of nonhomologous end-joining(NHEJ) pathway of double-stranded break repair(DSBR). Although it was reported that apurinic aprimidinic endonuclease 1(APE1) could promote the expression and activity of accumulated DSBR related proteins through regulating the gene transcription or posttranslational modification in order to maintain the chromosome integrity and genomic stability, the precise mechanism toward telomere maintenance remains unclear. Here we demonstrate that APE1 interacts with and promotes DNA-PKcs kinase activity. Furthermore, there is an increase association between hn RNPA1 and DNA-PKcs and hn RNPA1 phosphorylation by DNA-PKcs in vivo during G2 and M phases, and then DNA-PKcs dependent hn RNPA1 phosphorylation could promote the RPA-to-POT1 switch at single-stranded telomeric DNA. Consequently, cells lacking hn RNPA1 or DNA-PKcs dependent modification lead to significant sister telomere fusions. Taken together, our results indicate that the regulatory pathway of “APE1─ DNA-PKcs─ hn RNPA1” is critical for formation of the protective capping structure at the newly replicated telomeres to prevent the accumulation of telomeric aberrations.Objective1. To investigate the molecular mechanism of APE1 redox function in regulating DNA-PKcs kinase activity;2. To investigate the regualtory effect of DNA-PKcs on hn RNPA1 phosphorylation during mitosis;3. To investigate the regualtory effect of phosphorylated hn RNPA1 on promoting RPA-to-POT1 switch at single-stranded telomeric DNA and its protective effect on mitotic telomere.Materials and Methods1. The influence of APE1 redox function in DNA-PKcs kinase activity: Applying the methods of western blot, immunofluorescence, DNA end joining activity and coimmunoprecipitation to test APE1 knockdown and its redox deficient cell lines, aiming to investigate the regulatory effect of APE1 and its redox function on DNA-PKcs preotein level, kinase activity and NHEJ repair function.2. The research on the mechanism of DNA-PKcs mediated mitotic hn RNPA1 phosphorylation: Using the meterials of antibody for substrate phosphorylation, specific kinase inhibitors, site- directed mutagenesis and purified proteins, and applying the methods of cell cycle synchronization, western blot, co-immunoprecipitation, GST- pulldown to unveil the interacting domain between DNA-PKcs and hn RNPA1, and the influence factors on DNA-PKcs mediated hn RNPA1 phosphorylation.3. The protective effect of phosphorylated hn RNPA1 on mitotic telomere: Using the meterials of specific kinase inhibitors, synthetic single-stranded telomeric DNA and PNA probes to test hn RNPA1 knockdown and its functional mutant cell lines, aiming to investigate the regualtory effect of phosphorylated hn RNPA1 on promoting RPA-to-POT1 switch at single-stranded telomeric DNA and its protective effect on mitotic telomere.Results1. The influence of APE1 redox function in DNA-PKcs kinase activity: APE1 and DNA-PKcs could form a protein complex, and APE1 redox function could regulate DNAPKcs kinase activity and its NHEJ repair function, instead of DNA-PKcs protein level.2. The research on the mechanism of DNA-PKcs mediated mitotic hn RNPA1 phosphorylation: The interaction between DNA-PKcs and hn RNP-A1 during G2/M phases facilitates robust phosphorylation of hn RNPA1 at Ser95 and Ser192; The domain mapping showed that the PRD domain in the minimum region of DNA-PKcs interacts with hn RNP-A1, and the N-terminal RRMs contribute to the direct protein-protein interaction between hn RNP-A1 and DNA-PKcs; Further, single-stranded telomeric DNA and DNAPKcs compete for the binding of hn RNPA1.3. The protective effect of phosphorylated hn RNPA1 on mitotic telomere: Phosphomimetic mutants of hn RNPA1, with increased affinity to single-stranded telomeric DNA, were able to support the loading of POT1 to single-stranded telomeric DNA better than non-phospho hn RNP-A1 mutants; DNA-PKcs and its kinase activity could facilitate the affinity of hn RNPA1 to single-stranded telomeric DNA and its RPA displacement activity; Phosphomimetic mutants of hn RNP-A1 can restore RPA displacing activity in DNA-PKcs defective cells; Further, hn RNP-A1 and its phosphorylation are required for prevention of DDR at telomere during mitosis; Consistently, T-FISH analysis revealed that significant aberrations were found in hn RNP-A1 deficient cells and cells expressing non-phospho hn RNP-A1 mutants including telomere-free chromosome ends and sister telomere fusions.Conclusion1. Multi-functional APE1 protein regulates DNA-PKcs kinase activity and its repair function through interacting with DNA-PKcs, which is dependent on APE1 redox function.2. The high affinity of DNA-PKcs to hn RNP-A1 during mitosis was observed to be through PRD domain of DNA-PKcs and N-terminal RRMs of hn RNPA1.3. DNA-PKcs phosphorylates hn RNPA1 at Ser95 and Ser192 within its RRMs during mitosis.4. Phosphomimetic mutations augmented hn RNP-A1 binding to single-stranded telomeric DNA whereas non-phospho hn RNP-A1 mutants decreased hn RNP-A1 binding to single-stranded telomeric DNA during mitosis.5. Phosphorylated hn RNPA1 promotes the RPA-to-POT1 switch at single-stranded telomeric DNA with high efficiency during mitosis, thus preventing ATR-mediated DDR at telomeres.6. The role of hn RNP-A1 in the RPA-to-POT1 switch is critical for telomere protection and maintenance of telomere integrity.