Development of a highly efficient method to integrate genes into genome of higher eukaryotic cells by homologous recombination

开发一种通过同源重组将基因整合到高等真核细胞基因组中的高效方法

• 批准号: 12557208
• 负责人: ENOMOTO Takemi
• 金额: $ 7.62万
• 依托单位: Tohoku University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2000
• 资助国家: 日本
• 起止时间: 2000 至 2001
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-12557208/
• 关键词: Bloom syndrome; homologous recobination; targeted integration; BLM; ATM; yeast Sgs1; RecQL1; RecQL5; RecQファミリーヘリカーゼ; targeted-integration; 阻害剤スクリーニング

The aim of this study is to identify the proteins involved in the homologous recombination that is suppressed by BLM and to establish a method that enable us to perform targeted integration highly efficiently by inhibiting BLM function. Although we could not succeed to find good inhibitors for BLM, we obtained important information concerning the function of BLM and the recombination that occurs in the absence of BLM.1. By generating and analyzing double gene knockout DT40 cells, BLM and RAD54, RAD51, ATM, MRE11, RECQL1, or RECQL5, it was indicated that that the majority of increased sister chromatid exchanges (SCEs) in BLM disrupted cells are formed via homologous recombination, and the contribution of ATM and MRE 11 to the SCE is relatively small. In contrast, ATM greatly contributed to the targeted integration the frequency of which was increased by disruption of BLM gene.2. The analysis using DT 40 cells also indicated that RecQL5 was involved in the targeted integration in BLM gene knockout cells.3. An analysis of yeast SGS1 (the yeast homologue of BLM) gene disruptants indicated that Sgs1 functions to suppress recombination under normal conditions but functions to induce recombination under damage-inducing conditions probably interacting with topoisomerase III.

本研究的目的是鉴定参与 BLM 抑制的同源重组的蛋白质，并建立一种方法，使我们能够通过抑制 BLM 功能来高效地进行靶向整合。尽管我们未能成功找到良好的 BLM 抑制剂，但我们获得了有关 BLM 功能以及在 BLM.1 缺失时发生的重组的重要信息。通过生成和分析双基因敲除DT40细胞、BLM和RAD54、RAD51、ATM、MRE11、RECQL1或RECQL5，表明BLM破坏细胞中增加的姐妹染色单体交换（SCE）大部分是通过同源重组形成的，ATM和MRE 11对SCE的贡献相对较小。相比之下，ATM对靶向整合有很大贡献，并且通过破坏BLM基因可以增加靶向整合的频率。2． DT 40细胞分析也表明RecQL5参与了BLM基因敲除细胞的靶向整合。 3．对酵母 SGS1（BLM 的酵母同源物）基因破坏体的分析表明，Sgs1 在正常条件下发挥抑制重组的作用，但在损伤诱导条件下发挥诱导重组的作用，可能与拓扑异构酶 III 相互作用。

项目成果

Takemi Enomoto: "Functions of RecQ family helicases : possible involvement of Bloom's and Werner's syndrome gene products in guarding genome integrity during DNA replication"Journal of Biochemistry. 129. 501-507 (2001)

Takemi Enomoto：“RecQ 家族解旋酶的功能：布卢姆综合征和沃纳综合征基因产物可能参与 DNA 复制过程中保护基因组完整性”《生物化学杂志》。

Ayako Ui: "Possible involvement of Top3 via the N-terminal region of Sgs1 in the complementation of MMS sensitivity and the suppression of hyper recombination."Molecular General Genetics. (in press).

Ayako Ui：“Top3 可能通过 Sgs1 的 N 末端区域参与 MMS 敏感性的补充和超重组的抑制。”《分子普通遗传学》。

Atsuko Miyajima: "Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions."Mol. Cell. Biol.. 20. 6399-6409 (2000)

Atsuko Miyajima：“Sgs1 解旋酶活性是有丝分裂所必需的，但显然不是减数分裂功能所必需的。”Mol.

Yuriko Hachiya: "Immunohistochemical expression and pathogenesis of BLM in the human brain and visceral organs."Neuropathol.. 21. 123-128 (2001)

Yuriko Hachiya：“人脑和内脏器官中 BLM 的免疫组织化学表达和发病机制。”Neuropathol.. 21. 123-128 (2001)

Atsuko Miyajima: "Sgs1 helicase activity is required for mitotic but apparently not for meiotic functions"Molecular Cellular Biology. 20. 6399-6409 (2000)

Atsuko Miyajima：“Sgs1 解旋酶活性是有丝分裂所必需的，但显然不是减数分裂功能所必需的”分子细胞生物学。