DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells. Biochem.
DNA-PK inhibition causes a low level of H2AX phosphorylation and homologous recombination repair in Medaka (Oryzias latipes) cells. Biochem.
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DNA-PK 抑制会导致青鳉 (Oryzias latipes) 细胞中 H2AX 磷酸化和同源重组修复水平降低。
DOI：
10.1016/j.bbrc.2012.10.128
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发表时间：
2012
期刊：
影响因子：
--
通讯作者：
H.
中科院分区：
文献类型：
--
作者：
Urushihara;Y.;Kobayashi;J.;Matsumoto;Y.;Komatsu;K;Oda;S.;Mitani;H.
非同源末端连接(NHEJ)和同源重组(HR)被称为DNA双链断裂(DSB)修复途径。已有研究表明，PI3蛋白家族成员DNA-PK促进NHEJ的发生，DNA-PK异常导致NHEJ缺陷。然而，在这项研究中，我们证明了用DNA-PK抑制剂处理的野生型细胞系和DNA-PK功能异常的突变细胞系在鱼类细胞(Medaka，Oryzias Latipe)中表现出较低的HR效率。此前，我们报道了辐射敏感突变株RIC1在γ照射后组蛋白H_2AX的磷酸化缺陷。在这里，我们发现，DNA-PK抑制剂NU7026处理后，野生型细胞的γH_2AX焦点数量显著减少，但对γ细胞没有显著影响。此外，与野生型细胞相比，RIC1细胞的DNA-PK激酶活性显著降低。我们研究了DSB诱导后的NHEJ和HR效率。野生型细胞经NU7026和RIC1细胞处理后，HR效率降低。这些结果表明，DNA-PK基因的异常导致γH_2AX焦点数目减少和HR效率降低。γ照射后，我们进行磷酸化DNA-PKcs(Thr2609)和53BP1焦点分析。与野生型细胞相比，RIC1细胞的DNA-PKcs磷酸化节点数明显减少，53BP1节点数无明显差异。这些结果表明，低水平的DNA-PK活性导致RIC1细胞DNA-PKcs自磷酸化异常。已知53BP1参与DNA-PK依赖性和非依赖性NHEJ。因此，我们认为DNA-PK非依赖的NHEJ在DNA-PK活性降低的情况下修复DSB，从而导致HR效率降低。
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.