title: DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells.
abstract: Nonhomologous end joining (NHEJ) and homologous recombination (HR) are known as DNA double-strand break (DSB) repair pathways. It has been reported that DNA-PK, a member of PI3 kinase family, promotes NHEJ and aberrant DNA-PK causes NHEJ deficiency. However, in this study, we demonstrate that a wild-type cell line treated with DNA-PK inhibitor and a mutant cell line with dysfunctional DNA-PK showed decreased HR efficiency in fish cells (Medaka, Oryzias latipes). Previously, we reported that the radiation-sensitive mutant RIC1 strain has a defect in the Histone H2AX phosphorylation after γ-irradiation. Here, we showed that a DNA-PK inhibitor, NU7026, treatment resulted in significant reduction in the number of γH2AX foci after γ-irradiation in wild-type cells, but had no significant effect in RIC1 cells. In addition, RIC1 cells showed significantly lower levels of DNA-PK kinase activity compared with wild-type cells. We investigated NHEJ and HR efficiency after induction of DSBs. Wild-type cells treated with NU7026 and RIC1 cells showed decreased HR efficiency. These results indicated that aberrant DNA-PK causes the reduction in the number of γH2AX foci and HR efficiency in RIC1 cells. We performed phosphorylated DNA-PKcs (Thr2609) and 53BP1 focus assay after γ-irradiation. RIC1 cells showed significant reduction in the number of phosphorylated DNA-PKcs foci and no deference in the number of 53BP1 foci compared with wild-type cells. These results suggest that low level of DNA-PK activity causes aberrant DNA-PKcs autophosphorylation in RIC1 cells. It is known that 53BP1 is involved in both DNA-PK dependent and independent NHEJ. Therefore we suggest that DNA-PK independent NHEJ repair DSBs under the condition of decreased DNA-PK activity, which causes reduction of HR efficiency.
authors.affiliation: Department of Integrated Biosciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba 277-8562, Japan.
authors.forename: Yusuke
authors.initials: Y
authors.lastname: Urushihara
authors.forename: Junya
authors.initials: J
authors.lastname: Kobayashi
authors.forename: Yoshihisa
authors.initials: Y
authors.lastname: Matsumoto
authors.forename: Kenshi
authors.initials: K
authors.lastname: Komatsu
authors.forename: Shoji
authors.initials: S
authors.lastname: Oda
authors.forename: Hiroshi
authors.initials: H
authors.lastname: Mitani
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002867:Chromones / Q000494:pharmacology; D053903:DNA Breaks, Double-Stranded; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D005720:Gamma Rays; D017930:Genes, Reporter; D006657:Histones / Q000378:metabolism*; D008969:Molecular Sequence Data; D009025:Morpholines / Q000494:pharmacology; D009990:Oryzias / Q000235:genetics* / Q000378:metabolism*; D010766:Phosphorylation; D059767:Recombinational DNA Repair; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism
chemical_list: C479235:2-(morpholin-4-yl)benzo(h)chromen-4-one; D002867:Chromones; D006657:Histones; D009025:Morpholines; D016159:Tumor Suppressor Protein p53; D051747:DNA-Activated Protein Kinase