Analysis of protein kinase U (PKU) that may be recruited to the DNA double-strand breaks

分析可能被招募至 DNA 双链断裂的蛋白激酶 U (PKU)

• 批准号: 14572146
• 负责人: DATE Takayasu
• 金额: $ 1.92万
• 依托单位: KANAZAWA MEDICAL UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2002
• 资助国家: 日本
• 起止时间: 2002 至 2003
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-14572146/
• 关键词: PKU-beta; TLK1; 53BP1; X-ray; DNA double-strand break; DNA repair; siRNA; chromosomal segregation; centrosome; TLK-2; クロマチン; PKU; IRIF; RNAi; PKU結合タンパク質

1)X-ray irradiation induced foci (IRIF) was observed in nuclei of HeLa or MCF7 cells when cells were immunostained by polyclonal anti-PKU-β (TLK1) antibodies. However, IRIF was not detected by monoclonal antibody. There is no significant difference in reacted proteins by two antibodies. Two of three proteins that recognized by antibodies in MCF7 cell lysate were identified as splicing variants.2)When exogenous HA-tagged PKU was introduced into cells, no IRIF was observed by anti-Ha antibody. However, we could not determine whether failure was due to overexpression.3)The siRNA significantly reduced the expression of PKU-beta at 3 days after transfedction. These cells, howver, still formed IRIF.4)MCF7 cells with low expression of PKU-beta mediated by siRNA was sensitive to either X-ray irradiation, UV-irradiation or genotoxic reagents such as mytomisin C.5)When analyzed by FACS, PKU-knockdown with MCF 7 cells lead to aneuploidy or polyploidy. The number of chromosome of MCF7 which usually contains 62 increased to 62-120 at three days after the siRNA transfection.6)RNAi-mediated disruption of PKU-beta in cells interferes with normal spindle assembly and positioning. SiRNA treatment blocked the separation and positioning of duplicated centrosomes, thereby preventing the migration of the microtubule asters to opposite sides of chromosomes. These results suggests that PKU-beta involves G2-M transition and plays an important role on chromosomal segregation.

1）用多克隆抗PKU-β（TLK 1）抗体免疫组化染色，在HeLa和MCF 7细胞核内观察到X射线照射诱导的病灶（IRIF）。然而，IRIF未被单克隆抗体检测到。两种抗体反应的蛋白质没有显着差异。2）外源HA标记的PKU导入MCF 7细胞后，抗HA抗体检测不到IRIF的表达。然而，我们无法确定失败是否是由于过表达造成的。3）转染后3天，siRNA显着降低了PKU-β的表达。然而，这些细胞仍然形成IRIF。4）具有由siRNA介导的PKU-β低表达的MCF 7细胞对X射线照射、UV照射或遗传毒性试剂（如mytomisin C）敏感。5）当通过FACS分析时，用MCF 7细胞敲除PKU导致非整倍体或多倍体。MCF 7的染色体数目在siRNA转染后3天增加到62-120。6）RNAi介导的PKU-β在细胞中的破坏干扰了正常的纺锤体组装和定位。SiRNA处理阻断了重复中心体的分离和定位，从而阻止了微管星形细胞向染色体相对侧的迁移。这些结果表明PKU-β参与了G2-M转换，并在染色体分离中起重要作用。

项目成果

Iwabuchi, K., Date, T et al.: "Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA."J. Biol. Chem.. 278. 36487-36495

Iwabuchi, K., Date, T 等人：“53BP1 通过与 DNA 直接相互作用在 DNA 末端连接修复中的潜在作用。”

Iwabuchi K, Date T et al.: "Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA."J Biol Chem.. 278・38. 36487-36495 (2003)

Iwabuchi K、Date T 等：“53BP1 通过与 DNA 直接相互作用在 DNA 末端连接修复中的潜在作用。”J Biol Chem. 278・36495 (2003)。

Iwabuchi K, Basu BP, Kysela B, Kurihara T, Shibata M, Guan D, Cao Y, Hamada T, Imamura K, Jeggo PA, Date T, Doherty AJ: "Potential role for 53BP1 in DNA end-joining repair through direct interaction with DNA."J.Biol.Chem.. 278. 36487-36495 (2003)

Iwabuchi K、Basu BP、Kysela B、Kurihara T、Shibata M、Guan D、Cao Y、Hamada T、Imamura K、Jeggo PA、Date T、Doherty AJ：“53BP1 通过直接相互作用在 DNA 末端连接修复中的潜在作用