Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA
Mismatch repair-dependent processing of methylation damage gives rise to persistent single-stranded gaps in newly replicated DNA
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DOI：
10.1101/gad.455407
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发表时间：
2007-12-15
影响因子：
10.5
通讯作者：
Jiricny, Josef
中科院分区：
文献类型：
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作者：
Mojas, Nina;Lopes, Massimo;Jiricny, Josef
O-6-甲基鸟嘌呤（O-6-Methylguanine，（Me）G）是由S（N）1型甲基化试剂产生的具有高度细胞毒性的DNA修饰。尽管许多研究表明DNA复制、错配修复（MMR）和同源重组（HR）与（Me）G毒性有关，但其作用模式仍然难以捉摸。本文研究了N-甲基-N ′-硝基-N-亚硝基胍（MNNG）处理酵母和哺乳动物细胞DNA后的分子变化。虽然在治疗后的第一个细胞周期中复制叉进展不受影响，但电子显微镜分析显示在新复制的DNA中积累了（Me）G和MMR依赖的单链DNA（ssDNA）缺口。进入第二个细胞周期需要HR，而随后的G（2）阻滞需要（Me）G的持续存在。酵母细胞克服了这个障碍，而哺乳动物细胞通常无法恢复，并且那些包含多个姐妹染色单体交换的细胞。值得注意的是，可以通过在第一个S相之后去除（Me）G来废除该阻滞。这些新的数据为MMR试图纠正复制过程中出现的（Me）G/C或（Me）G/T错配的假设提供了令人信服的支持。由于（Me）G在暴露的模板链中的持久性，修复合成不能发生，这在复制叉后面留下了单链缺口。在随后的S期，这些缺口导致复制叉崩溃，并引发重组和细胞周期停滞。
O-6-Methylguanine ((Me)G) is a highly cytotoxic DNA modification generated by S(N)1-type methylating agents. Despite numerous studies implicating DNA replication, mismatch repair (MMR), and homologous recombination (HR) in (Me)G toxicity, its mode of action has remained elusive. We studied the molecular transactions in the DNA of yeast and mammalian cells treated with N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Although replication fork progression was unaffected in the first cell cycle after treatment, electron microscopic analysis revealed an accumulation of (Me)G-and MMR-dependent single-stranded DNA (ssDNA) gaps in newly replicated DNA. Progression into the second cell cycle required HR, while the following G(2) arrest required the continued presence of (Me)G. Yeast cells overcame this block, while mammalian cells generally failed to recover, and those that did contained multiple sister chromatid exchanges. Notably, the arrest could be abolished by removal of (Me)G after the first S phase. These new data provide compelling support for the hypothesis that MMR attempts to correct (Me)G/C or (Me)G/T mispairs arising during replication. Due to the persistence of (Me)G in the exposed template strand, repair synthesis cannot take place, which leaves single-stranded gaps behind the replication fork. During the subsequent S phase, these gaps cause replication fork collapse and elicit recombination and cell cycle arrest.