Analysis of error-prone polymerases in vertebrate cells

脊椎动物细胞中易错聚合酶的分析

• 批准号: 15510049
• 负责人: SONODA Eiichiro
• 金额: $ 2.5万
• 依托单位: Kyoto University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for Scientific Research (C)
• 财政年份: 2003
• 资助国家: 日本
• 起止时间: 2003 至 2004
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-15510049/
• 关键词: DT40; TLS polymerases; Translesion DNA synthesis; homologous recombination; rad30; rev3; Cisplatin; Postreplication repair; 損傷乗り越え修復; 突然変異; 損傷乗り越えポリメラーゼ

Damaged DNA can be a serious threat to successful completion of DNA replication. If a replication fork encounters a damage on the template DNA it arrest, resulting in a large gap on daughter strand and occasionally a break. These lesions are processed by a mechanism called postreplication repair (PRR) that enables the DNA replication without removing the lesions. Two different repair pathways are thought to mediate PRR. Homologous recombination(HR) uses an undamaged sister duplex as a template to repair gaps and breaks. Translesion DNA synthesis (TLS), controlled by a putative E3 ubiquitine ligase Rad18 and E2 enzyme Rad6,relies on non-essential DNA polymerases such as polζ (Rev3/Rev7) and polη (Rad30) to synthesize DNA across the lesion. To study the molecular basis of PRR in vertebrates, we have established single and multiple knockout-mutants of Rev1,Rev3,Rev7,Rad18,Rad30,PolK,PolQ(involved in TLS) and Rad51 paralog and Rad54 (involved in HR) genes using chicken DT40 cells.HR defect … More ive mutants showed a decreased level of sister chromatid exchange (SCE) which reflects a DNA repair process by crossover-type HR and accumulated spontaneous chromosomal breaks. In contrast, TLS mutants including rev1,rev3,rev7, polκ and rad18 mutants showed a significant increase in SCE, suggesting that in the absence of TLS,HR predominates to bypass the spontaneous damages. rev and rad18 mutants were highly sensitive to a wide range of DNA-damaging agents, including UV, ionizing radiation, MMS and cross-linking agents. rev single mutants and rev1/3/7 triple mutant showed almost comparable sensitivities against various genotoxic stresses, suggesting that Rev1 and polζ (Rev3/7) function as a functional unit in DNA damage response. On the other hand, rad30 and polκ showed the sensitivity only to UV, suggesting that each TLS polymerase play a distinctive role in DNA damage response. Interestingly, rev1 and rad30 but not rev3 mutant exhibited a significant decrease in the frequency of immunoglobulin gene conversion, suggesting the participation of these molecules in a recombination-based pathway, such as DNA synthesis step of intragenic homologous recombination. Thus, the molecules involved in TLS pathway play a variety of roles in DNA repair and recombination. Less

受损的 DNA 可能对 DNA 复制的成功完成构成严重威胁，如果复制叉在模板 DNA 上遇到损伤，则会导致子链出现大间隙，有时还会出现断裂。这些损伤是通过一种称为复制后的机制来处理的。修复 (PRR) 可以在不去除损伤的情况下进行 DNA 复制。同源重组 (HR) 使用未受损的姐妹双链体作为模板来修复缺口和断裂。 （TLS）由假定的 E3 泛素连接酶 Rad18 和 E2 酶 Rad6 控制，依赖于非必需 DNA 聚合酶，如 pol z (Rev3/Rev7) 和 pol n (Rad30) 来跨病变合成 DNA，以研究其分子基础。在脊椎动物中，我们已经建立了单个和多个敲除突变体Rev1、Rev3、Rev7、Rad18、Rad30、PolK、PolQ（参与 TLS）和 Rad51 旁系同源物和 Rad54（参与 HR）基因使用鸡 DT40 细胞。HR 缺陷…更多 ive 突变体显示姐妹染色单体交换水平降低（SCE） ）反映了交叉型 HR 的 DNA 修复过程和累积的自发染色体断裂，相比之下，TLS 突变体包括。 rev1、rev3、rev7、polκ 和 rad18 突变体的 SCE 显着增加，表明在没有 TLS 的情况下，HR 主要绕过自发损伤，而 rev 和 rad18 突变体对多种 DNA 损伤剂高度敏感。包括紫外线、电离辐射、MMS 和交联剂 rev 单突变体和 rev1/3/7 三重突变体对各种基因毒性胁迫表现出几乎相当的敏感性，表明 Rev1 和 pol z (Rev3/7) 在 DNA 损伤反应中发挥功能单位的作用，而 rad30 和 polκ 仅对 UV 敏感，表明每种 TLS 聚合酶在 DNA 损伤反应中发挥着独特的作用。 rev1 和 rad30 但不是 rev3 突变体表现出免疫球蛋白基因转换频率显着降低，表明这些分子参与基于重组的途径，例如基因内的 DNA 合成步骤因此，参与 TLS 途径的分子在 DNA 修复和重组中发挥多种作用。

项目成果

Morrison, C., Vagnarelli, P., Sonoda, E., Takeda, S., Earnshaw, W.C.: "Sister chromatid cohesion and genome stability in vertebrate cells"Biochem Soc Trans. 31. 263-265 (2003)

Morrison, C.、Vagnarelli, P.、Sonoda, E.、Takeda, S.、Earnshaw, W.C.：“脊椎动物细胞中的姐妹染色单体内聚力和基因组稳定性”Biochem Soc Trans。

Similar effects of Brca2 truncation and Rad51 paralog deficiency on immunoglobulin V gene diversification in DT40 cells support an early role for

Brca2 截短和 Rad51 旁系同源物缺陷对 DT40 细胞中免疫球蛋白 V 基因多样化的类似影响支持了

• 发表时间: 2005
• 期刊: Mol Cell Biol 25
• 作者: Hatanaka A;Yamazoe M;Sale JE;Takata M;Yamamto K;Kitao H;Sonoda E et al.
• 通讯作者: Sonoda E et al.

Post-replication repair in DT40 cells : translesion polymerases versus recombinases.

DT40 细胞的复制后修复：跨损伤聚合酶与重组酶。

• 发表时间: 2004
• 期刊: Bioessays 26
• 作者: Hochegger;H.;Sonoda;E.;Takeda;S.
• 通讯作者: S.

Dynamic control of Rad51 recombinase by self-association and interaction with BRCA2.

• DOI: 10.1016/s1097-2765(03)00394-0
• 发表时间: 2003-10
• 期刊: Molecular cell
• 影响因子: 16
• 作者: David S. Yu;E. Sonoda;S. Takeda;C. L. Huang;L. Pellegrini;T. Blundell;A. Venkitaraman

Hochegger, H., Sonoda E., Takeda, S.: "Post-replication repair in DT40 cells : translesion polymerases versus recombinases"Bioessays. 26. 151-158 (2004)

Hochegger, H.、Sonoda E.、Takeda, S.：“DT40 细胞的复制后修复：跨损伤聚合酶与重组酶”生物论文。