DNA mismatch repair-dependent response to fluoropyrimidine-generated damage
DNA mismatch repair-dependent response to fluoropyrimidine-generated damage
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DOI：
10.1074/jbc.m412105200
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发表时间：
2005-02-18
影响因子：
4.8
通讯作者：
Boothman, DA
中科院分区：
文献类型：
--
作者：
Meyers, M;Wagner, MW;Boothman, DA
我们实验室以前的研究表明，MLH 1 DNA错配修复（AMR）基因的表达是在用5-氟-2 '-脱氧尿苷（FdUrd）处理后恢复HCT 116人结肠癌细胞以及Mlhl（-/-）鼠胚胎成纤维细胞的细胞毒性和有效的G1阻滞所必需的。在这里，我们发现，当MSH 2，另一个主要的AMM基因的表达，在HEC 59人子宫内膜癌细胞中恢复或存在于腺病毒E1 A永生化的Msh（+/+）（与同基因Msh 2（-/-））小鼠胚胎干细胞中时，发生了相同的现象。由于NIAM状态对胸苷酸合成酶抑制剂Tomudex的细胞反应（即G2期阻滞和致死率）几乎没有影响，并且在MMR缺陷细胞中发现了更高水平的[H-3]FdUrd掺入DNA，因此我们得出结论，ADM感受态细胞和MMR缺陷细胞之间的差异FdUrd细胞毒性是在DNA掺入水平介导的。ATP酶激活分析表明，hMSH 2-hMSH 6异二聚体仅识别FdUrd部分（作为DNA中的碱基5-氟尿嘧啶（FU））时，与鸟嘌呤错配，但不与腺嘌呤配对。此外，使用甲基-CpG结合结构域4糖基化酶分析掺入的FdUrd表明，在AUVIR缺陷的HCT 116细胞的DNA中存在更多的错误掺入的FU：Gua。我们的数据首次证明MMR特异性检测FU：Gua（在第一轮DNA复制中），发出持续G，逮捕和致死的信号。
Previous studies from our laboratory indicated that expression of the MLH1 DNA mismatch repair (AMR) gene was necessary to restore cytotoxicity and an efficient G, arrest in HCT116 human colon cancer cells, as well as Mlhl(-/-) murine embryonic fibroblasts, after treatment with 5-fluoro-2'-deoxyuridine (FdUrd). Here, we show that an identical phenomenon occurred when expression of MSH2, the other major AMM gene, was restored in HEC59 human endometrial carcinoma cells or was present in adenovirus E1A-immortalized Msh(+/+) (compared with isogenic Msh2(-/-)) murine embryonic stem cells. Because NIAM status had little effect on cellular responses (i.e. G2 arrest and lethality) to the thymidylate synthase inhibitor, Tomudex, and a greater level of [H-3]FdUrd incorporation into DNA was found in MMR-deficient cells, we concluded that the differential FdUrd cytotoxicity between ADM-competent and MMR-deficient cells was mediated at the level of DNA incorporation. Analyses of ATPase activation suggested that the hMSH2-hMSH6 heterodimer only recognized FdUrd moieties (as the base 5-fluorouracil (FU) in DNA) when mispaired with guanine, but not paired with adenine. Furthermore, analyses of incorporated FdUrd using methyl-CpG-binding domain 4 glycosylase indicated that there was more misincorporated FU:Gua in the DNA of AUVIR-deficient HCT116 cells. Our data provide the first demonstration that MMR specifically detects FU:Gua (in the first round of DNA replication), signaling a sustained G, arrest and lethality.