Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.
Crosstalk between repair pathways elicits double-strand breaks in alkylated DNA and implications for the action of temozolomide.
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DOI：
10.7554/elife.69544
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发表时间：
2021-07-08
期刊：
影响因子：
7.7
通讯作者：
Fujii S
中科院分区：
文献类型：
--
作者：
Fuchs RP;Isogawa A;Paulo JA;Onizuka K;Takahashi T;Amunugama R;Duxin JP;Fujii S
替莫唑胺（TMZ）是一种DNA甲基化剂，是胶质母细胞瘤治疗中使用的主要化疗药物。TMZ主要诱导N-烷基化加合物（N7-甲基鸟嘌呤和N3-甲基腺嘌呤）和一些O 6-甲基鸟嘌呤（O 6 mG）加合物。目前的模型提出，在DNA复制过程中，胸腺嘧啶被纳入O 6 mG，促进无效的错配修复循环（MMR），导致DNA双链断裂（DSB）。为了重新审视O 6 mG加工的机制，我们将质粒DNA与替莫唑胺模拟物N-甲基-N-亚硝基脲（MNU）反应，并将其在非洲爪蟾卵提取物中孵育。我们已经表明，在这个系统中，MMR蛋白富集在MNU处理的DNA上，我们观察到了强大的，MMR依赖的修复合成。我们的证据还表明，MMR，在O 6 mG：C网站启动，强烈刺激顺式修复处理的其他病变，如N-烷基化加合物。重要的是，MNU处理的质粒在提取物中显示DSB，其频率随烷基化剂量的平方线性增加。我们认为，DSB的结果从两个独立的修复过程中，一个涉及MMR在O 6 mG：C网站和其他涉及在附近的N-烷基化加合物的碱基切除修复。我们提出了一个新的，复制独立的作用机制TMZ，除了充分研究的细胞周期依赖的作用模式。
Temozolomide (TMZ), a DNA methylating agent, is the primary chemotherapeutic drug used in glioblastoma treatment. TMZ induces mostly N-alkylation adducts (N7-methylguanine and N3-methyladenine) and some O6-methylguanine (O6mG) adducts. Current models propose that during DNA replication, thymine is incorporated across from O6mG, promoting a futile cycle of mismatch repair (MMR) that leads to DNA double-strand breaks (DSBs). To revisit the mechanism of O6mG processing, we reacted plasmid DNA with N-methyl-N-nitrosourea (MNU), a temozolomide mimic, and incubated it in Xenopus egg-derived extracts. We have shown that in this system, MMR proteins are enriched on MNU-treated DNA and we observed robust, MMR-dependent, repair synthesis. Our evidence also suggests that MMR, initiated at O6mG:C sites, is strongly stimulated in cis by repair processing of other lesions, such as N-alkylation adducts. Importantly, MNU-treated plasmids display DSBs in extracts, the frequency of which increases linearly with the square of alkylation dose. We suggest that DSBs result from two independent repair processes, one involving MMR at O6mG:C sites and the other involving base excision repair acting at a nearby N-alkylation adduct. We propose a new, replication-independent mechanism of action of TMZ, which operates in addition to the well-studied cell cycle-dependent mode of action.