PCNA-MutSα-mediated binding of MutLα to replicative DNA with mismatched bases to induce apoptosis in human cells
PCNA-MutSα-mediated binding of MutLα to replicative DNA with mismatched bases to induce apoptosis in human cells
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DOI：
10.1093/nar/gki878
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发表时间：
2005-01-01
影响因子：
14.9
通讯作者：
Sekiguchi, M
中科院分区：
文献类型：
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作者：
Hidaka, M;Takagi, Y;Sekiguchi, M
修饰的碱基，如O-6-甲基鸟嘌呤，在暴露于烷化剂的细胞中产生并引起细胞凋亡。在用N-甲基-N-亚硝基脲处理的人细胞中，我们通过使用染色质提取物的免疫沉淀方法检测到由MutS α、MutL α和PCNA组成的蛋白质复合物在受损DNA上，其中蛋白质-蛋白质相互作用通过化学交联稳定。时程实验表明，由MSH 2和MSH 6蛋白组成的MutS α和PCNA与DNA结合形成初始复合物，而由MLH 1和PMS 2组成的MutL α在DNA受损时与复合物结合。这种连续的结合模式通过以下发现进一步证实：即使在缺乏MLH 1的细胞中也观察到PCNA-MutS α复合物在染色质上的缔合，而在不存在MSH 2的情况下，没有实现MutL α与染色质的缔合。此外，通过小干扰RNA减少PCNA含量或通过DNA聚合酶抑制剂aphidicolin抑制DNA复制，显著降低了PCNA-MutS α-MutL α复合物的水平，并且还抑制了caspase-3活性的增加，这是诱导细胞凋亡的标志。这些观察结果表明，细胞凋亡的诱导是通过PCNA的作用与DNA复制的进展相结合的。
Modified bases, such as O-6-methylguanines, are produced in cells exposed to alkylating agents and cause apoptosis. In human cells treated with N-methyl-N-nitrosourea, we detected a protein complex composed of MutS alpha, MutL alpha and PCNA on damaged DNA by immunoprecipitation method using chromatin extracts, in which protein-protein interactions were stabilized by chemical crosslinking. Time course experiments revealed that MutS alpha, consisting of MSH2 and MSH6 proteins, and PCNA bind to DNA to form an initial complex, and MutL alpha, composed of MLH1 and PMS2, binds to the complex when the DNA is damaged. This sequential mode of binding was further confirmed by the findings that the association of PCNA-MutS alpha complex on chromatin was observed even in the cells that lack MLH1, whereas in the absence of MSH2 no association of MutL alpha with the chromatin was achieved. Moreover, reduction in the PCNA content by small-interfering RNA or inhibition of DNA replication by aphidicolin, an inhibitor of DNA polymerase, significantly reduced the levels of the PCNA-MutS alpha-MutL alpha complex and also suppressed an increase in the caspase-3 activity, a hallmark for the induction of apoptosis. These observations imply that the induction of apoptosis is coupled with the progression of DNA replication through the action of PCNA.