A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides

预防鸟嘌呤核苷酸氧化引起突变的新机制

• 批准号: 14580687
• 负责人: TAKAGI Yasumitsu
• 金额: $ 2.3万
• 依托单位: Fukuoka Dental College
• 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-14580687/
• 关键词: DNA repair; DNA oxidation; Spontaneous mutation; oxidation of guanine nucleotides; Spontaneous carcinogenesis

MutT-related proteins degrade 8-oxo-7,8-dihydrodeoxyguanosine triphosphate (8-oxo-dGTP), a mutagenic substrate for DNA synthesis, in the nucleotide pool, thereby preventing DNA replication errors. During a search of GenBank EST database, we found a new member of MutT-related protein, MTH2, which possesses the 23-amino acid MutT module. The cloned mouse MTH2 (mMTH2) cDNA was expressed in Escherichia coli mutT(-) cells and the protein was purified. mMTH2 protein hydrolyzes 8-oxo-dGTP to 8-oxo-dGMP, with Km of 32&mgr;M. Expression of cDNA for mMTH2 reduced significantly the elevated level of spontaneous mutation frequency of E. coli mutT(-) cells. Thus, MTH2 has a potential to protect the genetic material from the untoward effects of endogenous oxygen radicals. MTH2 could act as an MTH1 redundancy factor.Also, we reported that human cells have another mechanism for cleaning up the nucleotide pool to ensure accurate DNA replication. The human Nudix type 5 (NUDT5) protein hydrolyses 8-oxo-dGDP to monophosphate with a K(m) of 0.77 microM, a value considerably lower than that for ADP sugars, which were originally identified as being substrates of NUDT5. NUDT5 hydrolyses 8-oxo-dGTP only at very low levels, but is able to substitute for MutT when it is defective. When NUDT5 is expressed in E. coli mutT(-) cells, the increased frequency of spontaneous mutations is decreased to normal levels. Considering the enzymatic parameters of MTH1 and NUDT5 for oxidized guanine nucleotides, NUDT5 might have a much greater role than MTH1 in preventing the occurrence of mutations that are caused by the misincorporation of 8-oxoguanine in human cells.

MutT相关蛋白降解核苷酸库中DNA合成的诱变底物8-氧代-7，8-二氢脱氧鸟苷三磷酸（8-氧代-dGTP），从而防止DNA复制错误。通过对GenBank EST数据库的检索，我们发现了一个新的MutT相关蛋白MTH 2，它具有23个氨基酸的MutT模块。将克隆的小鼠MTH 2（mMTH 2）cDNA在大肠杆菌mutT（-）细胞中表达并纯化蛋白。mMTH 2蛋白水解8-oxo-dGTP为8-oxo-dGMP，Km为32 M。mMTH 2 cDNA的表达显著降低了E. colimutT（-）细胞。因此，MTH 2具有保护遗传物质免受内源性氧自由基的不利影响的潜力。此外，我们还报道了人类细胞有另一种机制来清理核苷酸库，以确保准确的DNA复制。人NUDT 5型（NUDT 5）蛋白质将8-氧代-dGDP水解为单磷酸，K（m）为0.77 μ M，该值显著低于ADP糖的值，ADP糖最初被鉴定为NUDT 5的底物。NUDT 5仅以非常低的水平水解8-氧代-dGTP，但当MutT有缺陷时能够替代MutT。当NUDT 5在E. colimutT（-）细胞中，自发突变频率的增加降低到正常水平。考虑到MTH 1和NUDT 5对氧化鸟嘌呤核苷酸的酶促参数，NUDT 5在防止由8-氧代鸟嘌呤错误掺入人类细胞中引起的突变的发生方面可能具有比MTH 1大得多的作用。

项目成果

Takagi, Y., Takahashi, M., Sanada, M., Ito, R., Yamaizumi, M., Sekiguchi, M.: "Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis."DNA Repair. 2. 1135-1146 (2003)

Takagi, Y.、Takahashi, M.、Sanada, M.、Ito, R.、Yamaizumi, M.、Sekiguchi, M.：“MGMT 和 MLH1 蛋白在烷基化诱导的细胞凋亡和诱变中的作用。”DNA 修复。

Sanada, M., Takagi, Y., Ito, R., Sekiguchi, M.: "Killing and mutagenic actions of dacarbazine, a chemotherapuetic alkylating agent, on human and mouse cells : effects of Mgmt and Mlh1 mutations."DNA repair. In press. (2004)

Sanada, M.、Takagi, Y.、Ito, R.、Sekiguchi, M.：“化疗烷化剂达卡巴嗪对人类和小鼠细胞的杀伤和诱变作用：Mgmt 和 Mlh1 突变的影响。”DNA 修复。

Takagi, Y., et al.: "Roles of MCMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis"DNA repair. 2. 1135 (2003)

Takagi, Y., et al.：“MCMT 和 MLH1 蛋白在烷基化诱导的细胞凋亡和诱变中的作用”DNA 修复。

Takagi, Y., et al.: "Roles of MGMT and MLH1 proteins in alkylation-induced apoptosis and mutagenesis."DNA repair. 2. 1135 (2003)

Takagi, Y., et al.：“MGMT 和 MLH1 蛋白在烷基化诱导的细胞凋亡和突变中的作用。”DNA 修复。

Ishibashi, T., Sekiguchi, M.: "A novel mechanism for preventing mutations caused by oxidation of guanine nucleotides"EMBO Report. 4. 479 (2003)

Ishibashi, T., Sekiguchi, M.：“防止鸟嘌呤核苷酸氧化引起突变的新机制”EMBO 报告。