Lips J, Kaina B
Division of Applied Toxicology, Institute of Toxicology, University of Mainz, Obere Zahlbacher Street 67, D-55131, Mainz, Germany.
Mutat Res. 2001 Nov 1;487(1-2):59-66. doi: 10.1016/s0921-8777(01)00105-7.
Methylation at the O(6)-position of guanine (O(6)-MeG) by alkylating agents is efficiently removed by O(6)-methylguanine-DNA methyltransferase (MGMT), preventing from cytotoxic, mutagenic, clastogenic and carcinogenic effects of O(6)-MeG-inducing agents. If O(6)-MeG is not removed from DNA prior to replication, thymine will be incorporated instead of cytosine opposite the O(6)-MeG lesion. This mismatch is recognized and processed by mismatch repair (MMR) proteins which are known to be involved in triggering the cytotoxic and genotoxic response of cells upon methylation. In this work we addressed three open questions. (1) Is MGMT able to repair O(6)-MeG mispaired with thymine (O(6)-MeG/T)? (2) Do MMR proteins interfere with the repair of O(6)-MeG/T by MGMT? (3) Does MGMT show a protective effect if it is expressed after replication of DNA containing O(6)-MeG? Using an in vitro assay we show that oligonucleotides containing O(6)-MeG/T mismatches are as efficient as oligonucleotides containing O(6)-MeG/C in competing for MGMT repair activity, indicating that O(6)-MeG mispaired with thymine is still subject to repair by MGMT. The addition of MMR proteins from nuclear extracts, or of recombinant MutSalpha, to the in vitro repair assay did not affect the repair of O(6)-MeG/T lesions by MGMT. This indicates that the presence of MutSalpha still allows access of MGMT to O(6)-MeG/T lesions. To elucidate the protective effect of MGMT in the first and second replication cycle after N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) treatment, MGMT transfected CHO cells were synchronized and MGMT was inactivated by pulse-treatment with O(6)-benzylguanine (O(6)-BG). Thereafter, the recovered cells were treated with MNNG and subjected to clonogenic survival assays. Cells which expressed MGMT in the first and second cell cycle were more resistant than cells which expressed MGMT only in the second (post-treatment) cell cycle. Cells which did not express MGMT in both cell cycles were most sensitive. This indicates that repair of O(6)-MeG can occur both in the first and second cell cycle after alkylation protecting cells from the killing effect of the lesion.
烷基化剂使鸟嘌呤的O(6)位发生甲基化（O(6)-甲基鸟嘌呤，O(6)-MeG），O(6)-甲基鸟嘌呤-DNA甲基转移酶（MGMT）可有效去除这种甲基化，防止O(6)-MeG诱导剂产生细胞毒性、致突变性、断裂剂性和致癌性作用。如果在复制前O(6)-MeG未从DNA中去除，胸腺嘧啶将取代胞嘧啶掺入到与O(6)-MeG损伤相对的位置。这种错配会被错配修复（MMR）蛋白识别并处理，已知MMR蛋白参与触发细胞在甲基化后的细胞毒性和基因毒性反应。在这项工作中，我们解决了三个悬而未决的问题。（1）MGMT能否修复与胸腺嘧啶错配的O(6)-MeG（O(6)-MeG/T）？（2）MMR蛋白是否会干扰MGMT对O(6)-MeG/T的修复？（3）如果MGMT在含有O(6)-MeG的DNA复制后表达，它是否具有保护作用？通过体外试验，我们发现含有O(6)-MeG/T错配的寡核苷酸在竞争MGMT修复活性方面与含有O(6)-MeG/C的寡核苷酸一样有效，这表明与胸腺嘧啶错配的O(6)-MeG仍可被MGMT修复。在体外修复试验中添加核提取物中的MMR蛋白或重组MutSalpha，并不影响MGMT对O(6)-MeG/T损伤的修复。这表明MutSalpha的存在仍允许MGMT接近O(6)-MeG/T损伤。为了阐明MGMT在N-甲基-N'-硝基-N-亚硝基胍（MNNG）处理后的第一个和第二个复制周期中的保护作用，将转染了MGMT的CHO细胞同步化，并用O(6)-苄基鸟嘌呤（O(6)-BG）脉冲处理使MGMT失活。此后，将恢复的细胞用MNNG处理并进行克隆形成存活试验。在第一个和第二个细胞周期中表达MGMT的细胞比仅在第二个（处理后）细胞周期中表达MGMT的细胞更具抗性。在两个细胞周期中都不表达MGMT的细胞最敏感。这表明烷基化后，O(6)-MeG的修复可在第一个和第二个细胞周期中发生，从而保护细胞免受损伤的杀伤作用。
