Institute of Toxicology, Medical Center of the University Mainz, Obere Zahlbacher Str 67, D-55131 Mainz, Germany.
DNA Repair (Amst). 2010 Oct 5;9(10):1050-63. doi: 10.1016/j.dnarep.2010.07.005. Epub 2010 Aug 13.
DNA-methylating agents of the S(N)2 type target DNA mostly at ring nitrogens, producing predominantly N-methylated purines. These adducts are repaired by base excision repair (BER). Since defects in BER cause accumulation of DNA single-strand breaks (SSBs) and sensitize cells to the agents, it has been suggested that some of the lesions on their own or BER intermediates (e.g. apurinic sites) are cytotoxic, blocking DNA replication and inducing replication-mediated DNA double-strand breaks (DSBs). Here, we addressed the question of whether homologous recombination (HR) or non-homologous end-joining (NHEJ) or both are involved in the repair of DSBs formed following treatment of cells with methyl methanesulfonate (MMS). We show that HR defective cells (BRCA2, Rad51D and XRCC3 mutants) are dramatically more sensitive to MMS-induced DNA damage as measured by colony formation, apoptosis and chromosomal aberrations, while NHEJ defective cells (Ku80 and DNA-PK(CS) mutants) are only mildly sensitive to the killing, apoptosis-inducing and clastogenic effects of MMS. On the other hand, the HR mutants were almost completely refractory to the formation of sister chromatid exchanges (SCEs) following MMS treatment. Since DSBs are expected to be formed specifically in the S-phase, we assessed the formation and kinetics of repair of DSBs by γH2AX quantification in a cell cycle specific manner. In the cytotoxic dose range of MMS a significant amount of γH2AX foci was induced in S, but not G1- and G2-phase cells. A major fraction of γH2AX foci colocalized with 53BP1 and phosphorylated ATM, indicating they are representative of DSBs. DSB formation following MMS treatment was also demonstrated by the neutral comet assay. Repair kinetics revealed that HR mutants exhibit a significant delay in DSB repair, while NHEJ mutants completed S-phase specific DSB repair with a kinetic similar to the wildtype. Moreover, DNA-PKcs inhibition in HR mutants did not affect the repair kinetics after MMS treatment. Overall, the data indicate that agents producing N-alkylpurines in the DNA induce replication-dependent DSBs. Further, they show that HR is the major pathway of protection of cells against DSB formation, killing and genotoxicity following S(N)2-alkylating agents.
S(N)2 型 DNA-甲基化试剂主要靶向 DNA 的环氮，主要产生 N-甲基嘌呤。这些加合物通过碱基切除修复 (BER) 修复。由于 BER 缺陷会导致 DNA 单链断裂 (SSB) 的积累，并使细胞对这些试剂敏感，因此有人认为，某些损伤本身或 BER 中间产物（例如无嘌呤/嘧啶位点）具有细胞毒性，可阻断 DNA 复制并诱导复制介导的 DNA 双链断裂 (DSB)。在这里，我们探讨了同源重组 (HR) 或非同源末端连接 (NHEJ) 或两者是否都参与了用甲基甲磺酸 (MMS) 处理细胞后形成的 DSB 的修复。我们表明，HR 缺陷细胞（BRCA2、Rad51D 和 XRCC3 突变体）对 MMS 诱导的 DNA 损伤更为敏感，如集落形成、凋亡和染色体畸变所测量的，而 NHEJ 缺陷细胞（Ku80 和 DNA-PK(CS) 突变体）仅对 MMS 的杀伤、诱导凋亡和致染色体断裂效应敏感。另一方面，HR 突变体对 MMS 处理后姐妹染色单体交换 (SCE) 的形成几乎完全耐受。由于 DSB 预计会在 S 期特异性形成，我们通过 γH2AX 定量以细胞周期特异性方式评估 DSB 的形成和修复动力学。在 MMS 的细胞毒性剂量范围内，S 期细胞而非 G1 和 G2 期细胞中诱导了大量 γH2AX 焦点。大量 γH2AX 焦点与 53BP1 和磷酸化 ATM 共定位，表明它们是 DSB 的代表。用中性彗星测定法也证明了 MMS 处理后 DSB 的形成。修复动力学表明，HR 突变体在 DSB 修复中存在明显延迟，而 NHEJ 突变体以与野生型相似的动力学完成 S 期特异性 DSB 修复。此外，在 HR 突变体中抑制 DNA-PKcs 并不影响 MMS 处理后的修复动力学。总的来说，这些数据表明，在 DNA 中产生 N-烷基嘌呤的试剂会诱导复制依赖性 DSB。此外，它们表明 HR 是细胞对抗 S(N)2-烷基化试剂形成 DSB、杀伤和遗传毒性的主要保护途径。
