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

修饰的DNA碱基不能阻止复制分叉的进展，但会导致错误配对，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种系统，通过诱导细胞凋亡来消除具有这种错配的细胞。O^6-甲基鸟嘌呤(O^6 mg)介导的杀伤途径为揭示这种细胞凋亡防御系统提供了一个很好的模型。我们发现其中一个错配识别(MMR)蛋白的缺陷阻碍了细胞的凋亡，并增强了烷化剂的诱变作用，这表明这个识别系统在O^6 mg诱导的细胞凋亡中起着重要的作用。作为诱导这种凋亡的早期步骤，我们用免疫沉淀的方法在人类细胞损伤的DNA上检测到由MUTSA、MUTRA和PCNA组成的蛋白质复合体。时程实验表明，MutSA、(MSH2和MSH6)和PCNA与DNA结合形成初始复合体，当DNA损伤时，Mutla(MLH1和PMS2)与复合体结合。由于APAF-1在DNA损伤引起的细胞凋亡过程中被激活，从而阻断了DNA复制，因此了解APAF-1的激活是否也参与了O^6 mg引起的细胞凋亡是非常重要的。为了研究这一问题，我们构建了编码O^6 mg修复酶和/或APAF-1基因的mGMT基因缺陷小鼠，并建立了小鼠的细胞系(mGmt^&lt；-/-&gt；-/-&gt；Apaf1^&lt；-/-&gt；)。这些细胞系使我们发现APAF-1是这种凋亡所必需的，就像顺铂诱导的那样，它阻止了DNA的复制。MNU诱导途径的分支点和顺铂诱导途径的分支点似乎位于MMR系统和APAF-1之间。

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.