Analysis of Homologous DNA Recombination using the chicken DT40 B cell line

使用鸡 DT40 B 细胞系进行同源 DNA 重组分析

• 批准号: 17390076
• 负责人: TAKEDA Shunichi
• 金额: $ 9.09万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (B)
• 财政年份: 2005
• 资助国家: 日本
• 起止时间: 2005 至 2006
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-17390076/
• 关键词: Homologous Recombination; Targeted integration; DT40 cell; Gene targeting; Rad18; Ubc13; Fbh1; DNA polymerase; 遺伝学; 癌; 細胞・組織; 放射線; 老化

In higher eukaryotic cells, transfected DNA is occasionally integrated into the genome predominantly through random integration. A novel exception of this rule is chicken B lymphocyte lines, including DT40 cells, in which random integration and targeted one occur with similar frequencies. The goal of this project is to identify the molecular mechanisms underlying the efficient gene targeting in DT40 cells. While the mechanisms have not yet clarified, we have revealed a role of some factors in Homologous DNA Recombination (HR), as delineated below.(1) Yeast Rad18, E3 ubiquitin ligase is essential for postreplicational repair (PRR), but is not involved in HR. We found that Rad18 suppresses the toxic effect of nonhomolgous end-joining, and thereby facilitates HR (Saberi et al., 2007).(2) Ubc13, E3 ubiquitin ligase is involved in a type of PRR, but not in HR in lower eukaryotes. We found that Ubc13 plays a critical role in an initial step of HR dependent DSB repair (Zhao et al., 2007).(3) FBH1 seems to prevent aberrant HR by suppressing the assembly of Rad51 at DNA damage in the fission yeast. In DT40 cells, we found that FBH1 does not affect Rad51 accumulation, but acts in parallel with Bloom helicase to suppress HR at a late step (Kohzaki et al., 2007).

在高等真核细胞中，转染的 DNA 有时主要通过随机整合整合到基因组中。这一规则的一个新的例外是鸡 B 淋巴细胞系，包括 DT40 细胞，其中随机整合和靶向整合以相似的频率发生。该项目的目标是确定 DT40 细胞中有效基因靶向的分子机制。虽然机制尚未阐明，但我们已经揭示了同源 DNA 重组 (HR) 中某些因素的作用，如下所述。 (1) 酵母 Rad18、E3 泛素连接酶对于复制后修复 (PRR) 至关重要，但不参与其中在人力资源方面。我们发现Rad18抑制非同源末端连接的毒性作用，从而促进HR(Saberi et al., 2007)。(2) Ubc13、E3泛素连接酶参与一种PRR，但不参与低等真核生物的HR 。我们发现 Ubc13 在 HR 依赖性 DSB 修复的初始步骤中发挥着关键作用 (Zhao et al., 2007)。(3) FBH1 似乎通过抑制裂殖酵母中 DNA 损伤时 Rad51 的组装来防止异常 HR。在 DT40 细胞中，我们发现 FBH1 不影响 Rad51 积累，但与 Bloom 解旋酶并行作用，在后期抑制 HR (Kohzaki et al., 2007)。

项目成果

Cooperative Roles of Vertebrate Fbh1 and Blm DNA Helicases in Avoidance of Crossovers during Recombination Initiated by Replication Fork Collapse.

脊椎动物 Fbh1 和 Blm DNA 解旋酶在复制叉崩溃引发的重组过程中避免交叉的协同作用。

• 发表时间: 2007
• 作者: Kohzaki M; Hatanaka A; Sonoda E; Yamazoe M; Kikuchi K; Trung NV; Szuts D; Sale JE; Shinagawa H; Watanabe M; Takeda S
• 通讯作者: Takeda S

Differential usage of non-homologous end-joining and homologous recombination in double strand break repair.

非同源末端连接和同源重组在双链断裂修复中的差异应用。

• 发表时间: 2006
• 作者: Sonoda E; Hochegger H; Saberi A; Taniguchi Y; Takeda S
• 通讯作者: Takeda S

A critical role for the ubiquitin-conjugating enzyme Ubc13 in initiating homologous recombination.

泛素结合酶 Ubc13 在启动同源重组中发挥着关键作用。

• 发表时间: 2007
• 作者: Zhao GY; Sonoda E; Barber LJ; Oka H; Murakawa Y; Yamada K; Ikura T; Wang X; Kobayashi M; Yamamoto K; Boulton SJ; Takeda S
• 通讯作者: Takeda S

RAD18 and poly[ADP ribose]polymerase independently suppress the access of nonhomologous end joining to double strand breaks and facilitate homologous recombination mediated repair.

RAD18 和聚[ADP 核糖]聚合酶独立地抑制非同源末端连接至双链断裂并促进同源重组介导的修复。

• 发表时间: 2007
• 作者: Saberi A; Hochegger H; Szuts D; Lan L; Yasui A; Sale JE; Taniguchi Y; Murakawa Y; Hua W; Yokomori K; Helleday T; Teraoka H; Arakawa H; Buerstedde JM; Takeda S.
• 通讯作者: Takeda S.

Vertebrate POLQ and POL β cooperate in Base Excision Repair of Oxidative DNA Damage.

脊椎动物 POLQ 和 POL β 协同参与氧化 DNA 损伤的碱基切除修复。

• 发表时间: 2006
• 作者: Yoshimura A; Kohzaki M; Nakamura J; Asagoshi K; Sonoda E; Hou E; Prasad R; Wilson SH; Tano K; Yasui A; Lan L; Seki M; Wood RD; Arakawa H; Buerstedde JM; Hochegger H; Okada T; Hiraoka M; Takeda S
• 通讯作者: Takeda S