A High Throughput RNAi Screen Reveals the Protein Kinase NEK8 Regulates Replication Fork Protection via the DNA Repair Protein RAD51

Abstract

Proteins essential for homologous recombination play a pivotal role in the repair of DNA double strand breaks, DNA inter-strand crosslinks and replication fork stability. Defects in homologous recombination also play a critical role in the development of cancer and the sensitivity of these cancers to chemotherapy. RAD51, an essential factor for homologous recombination and replication fork protection, accumulates and forms immunocytochemically detectable nuclear foci at sites of DNA damage. To identify previously unknown kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 focus formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 focus formation. Interestingly, we found that knockout of Nek8 in murine embryonic fibroblasts leads to cellular sensitivity to the replication inhibitor, hydroxyurea (HU), but not to other DNA damaging agents. Furthermore, we show that NEK8 is required for proper replication fork protection following replication stall with HU. Loading of the essential DNA repair and replication fork protection factors, RAD51 and BRCA2, was decreased in NEK8 depleted and deficient cells lines. Using single-molecule DNA fiber analysis, we show that nascent DNA tracts are degraded in the absence of NEK8 following treatment with HU. We show an increase in genomic instability in Nek8 null murine cells following treatment with HU. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51. Lastly, the discovery that NEK8 functions in resistance to replication inhibiting agents raises the possibility that NEK8 may have clinical utility as a therapeutic target for tumor chemosensitization.