title: NEK8 regulates DNA damage-induced RAD51 foci formation and replication fork protection.
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 kinases that may regulate RAD51 localization to sites of DNA damage, we performed a human kinome siRNA library screen, using DNA damage-induced RAD51 foci formation as readout. We found that NEK8, a NIMA family kinase member, is required for efficient DNA damage-induced RAD51 foci formation. Interestingly, knockout of Nek8 in murine embryonic fibroblasts led to cellular sensitivity to the replication inhibitor, hydroxyurea, and inhibition of the ATR kinase. Furthermore, NEK8 was required for proper replication fork protection following replication stall with hydroxyurea. Loading of RAD51 to chromatin was decreased in NEK8-depleted cells and Nek8-knockout cells. Single-molecule DNA fiber analyses revealed that nascent DNA tracts were degraded in the absence of NEK8 following treatment with hydroxyurea. Consistent with this, Nek8-knockout cells showed increased chromosome breaks following treatment with hydroxyurea. Thus, NEK8 plays a critical role in replication fork stability through its regulation of the DNA repair and replication fork protection protein RAD51.
authors.affiliation: a Divisions of Human Biology and Public Health Sciences , Fred Hutchinson Cancer Research Center , Seattle , WA , USA.|b Molecular & Cellular Biology Graduate Program , University of Washington , Seattle , WA , USA.
authors.forename: Antonio
authors.initials: A
authors.lastname: Abeyta
authors.affiliation: a Divisions of Human Biology and Public Health Sciences , Fred Hutchinson Cancer Research Center , Seattle , WA , USA.
authors.forename: Maria
authors.initials: M
authors.lastname: Castella
authors.affiliation: a Divisions of Human Biology and Public Health Sciences , Fred Hutchinson Cancer Research Center , Seattle , WA , USA.
authors.forename: Celine
authors.initials: C
authors.lastname: Jacquemont
authors.affiliation: a Divisions of Human Biology and Public Health Sciences , Fred Hutchinson Cancer Research Center , Seattle , WA , USA.
authors.forename: Toshiyasu
authors.initials: T
authors.lastname: Taniguchi
mesh_terms: D004249:DNA Damage; D004261:DNA Replication; D042822:Genomic Instability; D006367:HeLa Cells; D059765:Homologous Recombination / Q000235:genetics; D006801:Humans; D000072379:NIMA-Related Kinases / Q000172:deficiency / Q000378:metabolism*; D034741:RNA, Small Interfering / Q000378:metabolism; D051135:Rad51 Recombinase / Q000378:metabolism*; D013312:Stress, Physiological
chemical_list: D034741:RNA, Small Interfering; C458529:NEK8 protein, human; D000072379:NIMA-Related Kinases; C499219:Nek8 protein, mouse; D051135:Rad51 Recombinase
keywords: DNA repair; NEK8; RAD51; genome instability; homologous recombination; replication fork protection