Kinase library screen identified VRK3 as a novel DNA damage factor

Abstract

DNA double-strand breaks (DSBs) are among the most cytotoxic DNA lesions that pose a significant threat to genome integrity. To counter DSBs, cells have evolved with a comprehensive signaling network collectively known as DNA damage response (DDR). DDR comprises of an intricate network of phosphorylation signaling events where ATM, ATR and DNA-PKcs act as master regulators. However, about 60% of the DSB-induced phosphorylation events are independent of these PIKKs and a vast majority of downstream DNA repair factors are devoid of the PIKK phosphorylation motif. The potential role of other kinases in orchestrating the transmission of DDR signals remains enigmatic. To systematically profile the involvement of kinases in DSB repair processes, we performed a CRISPR library screen that targets 715 essential human kinases. We have isolated vaccinia-related kinase 3 (VRK3) as a putative DNA damage factor that promotes DSB repair.

Consistent with a direct role in DSB repair, VRK3 localizes at laser-induced DNA damage tracks in a PARP-dependent manner. In addition, VRK3 inactivation not only leads to DSB repair defects marked by unresolved DNA damage foci, but also genomic instability evidenced from elevated frequencies of chromosomal aberrations. Accordingly, VRK3-associated DSB repair defect can be alleviated by reconstituting with wild type VRK3 but not its kinase-inactive K203E mutant. To further explore the underlying mechanism on how VRK3 facilitates DSB repair, analysis of the VRK3 interactome identified PARP1 and NHEJ machineries including Ku70/80 and DNA-PKcs as putative interacting partners. Notably, ablation of VRK3 leads to hyperaccumulation of Ku70/80 at DNA lesions during late S/G2 phase. Consistent with the speculation that VRK3 regulates the dynamic of these early repair machineries, cells depleted with VRK3 not only demonstrated impaired recruitment of RPA1/2 at laser damaged track but also defective IR-induced RPA1 foci formation. Importantly, compromised RPA loading in VRK3-deficient cells was alleviated upon inhibition of Ku70/80 binding on DNA and DNA-PK activation.

Analysis of VRK3-dependent phosphorylation events also uncovered upregulation of Ku70 on Ser520 where the phosphorylation was also enhanced during S/G2 phase. As S520A phospho-dead mutant of Ku70 demonstrated hyperaccumulation on DSBs, it is speculated that VRK1/VRK3-dependent phosphorylation of Ku70 on Ser520 may promote the dissociation of Ku70 from DSBs. However, the interactions among VRK1, VRK3 and Ku70 require further investigations.

Taken together, our findings have unveiled the role of VRK3 as a novel DNA damage factor that facilitates DSB repair by interacting with core NHEJ machineries.