DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells

Yusuke Urushihara, Junya Kobayashi, Yoshihisa Matsumoto, Kenshi Komatsu, Shoji Oda, Hiroshi Mitani

Research output: Contribution to journalArticlepeer-review

12 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)131-136
Number of pages6
JournalBiochemical and biophysical research communications
Volume429
Issue number3-4
DOIs
Publication statusPublished - 2012 Dec 14
Externally publishedYes

Keywords

  • 53BP1
  • DNA-PK
  • Double strand breaks (DSB)
  • H2AX
  • Medaka
  • NU7026

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

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