DHX9 phosphorylation at S321 by ATM regulates DHX9 retention at DNA double-strand break sites and interaction with BRCA1.

To preserve genome stability, the repair of DNA double-strand breaks (DSBs) that can be caused by exposure to ionizing radiation and certain anticancer drugs is of paramount importance. Recently, it became evident that various DNA-RNA helicases play a pivotal role in homologous recombination (HR) repair and non-homologous end joining, which are the two principal DSB repair machineries in mammalian cells. In a previous study, we reported that DHX9, which belongs to the DExH-box helicase family, is involved in HR repair. However, the regulatory mechanisms governing the function of DHX9 remains elusive. The present study has demonstrated that upon etoposide treatment, DHX9 was phosphorylated at S321 in a manner dependent on ataxia telangiectasia mutated (ATM), a protein kinase. In addition, cell cycle synchronization and fractionation analysis of cell extracts revealed that only chromatin-bound DHX9 was phosphorylated by ATM in the S phase, where HR repair functions. Furthermore, by live-cell imaging with unphosphorylated-mutant and phospho-mimic DHX9, we revealed that the S321 phosphorylation of DHX9 was required for the retention of DHX9 at DSB sites but not for the initial recruitment of DHX9 to DSB sites. The DSB repair efficiencies were found to be reduced in both cell lines expressing either the unphosphorylated mutant or the phospho-mimic DHX9. Consistent with this, phospho-mimic DHX9 showed reduced interaction with BRCA1. In conclusion, our findings indicate that the DSB-induced ATM-dependent phosphorylation of DHX9 at S321, which should be dynamically regulated, is crucial for efficiency of the DSB repair.