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The mechanism of cell death inducedby O^6-methylguanine DNA : the role of MSH2 and MLH1 proteins

O^6-甲基鸟嘌呤DNA诱导细胞死亡的机制：MSH2和MLH1蛋白的作用

基本信息

批准号：
16591902
负责人：
ITO Riyoko
金额：
$ 1.98万
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：
2004
资助国家：
日本
起止时间：
2004 至 2005
项目状态：
已结题

项目摘要

Modified DNA bases which do not prevent progression of the replication fork but cause mispairing are troublesome, since they yield mutation. To prevent such outcomes, multicellular organisms are equipped with a system to eliminate cells with such mispairs by inducing apoptosis. O^6-Methylguanine(O^6mG)-mediated killing pathway would provide an excellent model to reveal this apoptotic defense system. We have shown that defects in one of the mismatch recognition (MMR) proteins block apoptosis and enhance the mutagenic effects of alkylating agents, indicating the essential role of this recognition system in O^6mG-induced apoptosis.As an early step in the induction of this apoptosis, we detected a protein complex composed of MutSa, MutLa and PCNA on damaged DNA in human cells, by immunoprecipitation method. Time course experiments revealed that MutSa, (MSH2 and MSH6) and PCNA bind to the DNA to form an initial complex, and MutLa, (MLH1 and PMS2), binds to the complex when the DNA is damaged. These observations imply that the induction of this apoptosis is coupled with the progression of DNA replication through the action of PCNASince Apaf-1 is activated during apoptosis caused by DNA lesion that block DNA replication, it is important to know if the activation of Apaf-1 is also involved in apoptosis caused by O^6mG. To investigate this problem, we constructed mice defective in the Mgmt gene that encodes O^6mG repair enzyme and/or Apaf-1 gene, and established cell lines from the mice (Mgmt^<-/-> ; Mgmt^<-/->Apaf1^<-/->). These cell lines allowed us to find that Apaf-1 is required for this apoptosis, as in the case of the one induced by cisplatin, which blocks DNA replication. The branching point of the MNU-induced pathway and the one by cisplatin seems to exite somewhere between MMR system and Apaf-1.

修饰的DNA碱基不能阻止复制叉的进展，但会引起错配，这是很麻烦的，因为它们会产生突变。为了防止这种结果，多细胞生物体配备了一个系统，通过诱导凋亡来消除具有这种错配的细胞。O^6-甲基鸟嘌呤（O^6mG）介导的杀伤途径为揭示这种凋亡防御系统提供了一个很好的模型。我们已经发现，错配识别（MMR）蛋白中的一种缺陷会阻断细胞凋亡，并增强烷化剂的诱变效应，这表明该识别系统在O^6 mG诱导的细胞凋亡中起着重要作用。作为诱导细胞凋亡的早期步骤，我们通过免疫沉淀法检测到人类细胞受损DNA上的一种由MutSa、MutLa和PCNA组成的蛋白复合物。时程实验表明，MutSa（MSH 2和MSH 6）和PCNA结合到DNA上形成初始复合物，而MutLa（MLH 1和PMS 2）在DNA受损时结合到复合物上。这些观察结果表明，这种凋亡的诱导是通过PCNA的作用与DNA复制的进展相耦合的。由于Apaf-1在DNA损伤阻断DNA复制引起的凋亡过程中被激活，因此了解Apaf-1的激活是否也参与了O^6mG引起的凋亡是很重要的。为了研究这个问题，我们构建了编码0^6mG修复酶的Mgmt基因和/或Apaf-1基因缺陷的小鼠，并从小鼠建立了细胞系（Mgmt^<-/-> ; Mgmt^<-/-> Apaf ^<-/->）。这些细胞系使我们能够发现Apaf-1是这种细胞凋亡所必需的，就像顺铂诱导的细胞凋亡一样，顺铂可以阻断DNA复制。MNU诱导的通路和顺铂诱导的通路的分支点似乎位于MMR系统和Apaf-1之间。