Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage
Methylated DNA Causes a Physical Block to Replication Forks Independently of Damage Signalling, O6-Methylguanine or DNA Single-Strand Breaks and Results in DNA Damage
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DOI：
10.1016/j.jmb.2010.07.010
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发表时间：
2010-09-10
影响因子：
5.6
通讯作者：
Helleday, Thomas
中科院分区：
文献类型：
--
作者：
Groth, Petra;Auslander, Simon;Helleday, Thomas
尽管 DNA 烷基化剂在癌症治疗中已使用了数十年，但当复制叉遇到烷基化 DNA 时会发生什么仍不清楚。在这里，我们使用 DNA 纤维测定来研究烷化剂对复制叉进展的影响。我们发现烷基化剂甲磺酸甲酯（MMS）以剂量依赖性且与总体烷基化等级相关的方式抑制复制延伸。复制叉似乎被完全阻断，因为在 MMS 处理 1 小时后没有检测到核苷酸掺入。高剂量的 5 mM 咖啡因抑制大多数 DNA 损伤信号传导，总体上降低复制速率，但不会逆转 MMS 诱导的复制抑制，表明复制阻断与 DNA 损伤信号传导无关。此外，复制叉进展的受阻与 DNA 单链断裂的水平无关。 O-6-甲基鸟嘌呤 (O6meG)-DNA 甲基转移酶蛋白的过度表达负责去除毒性最强的烷基化 O6meG，但不会影响暴露于 N-甲基-M-硝基-N-亚硝基胍后的复制伸长。这表明 O6meG 损伤在哺乳动物细胞中被有效绕过。此外，我们发现 MMS 诱导的 γ H2AX 焦点与 53BP1 焦点和新复制区域共定位，表明 DNA 双链断裂是在 MMS 阻断的复制叉处形成的。总而言之，我们的数据表明，在接触烷基化剂期间形成的 N-烷基化会物理性地阻断哺乳动物细胞中的复制叉延伸，导致复制相关的 DNA 损伤的形成，可能是双链断裂。 (C) 2010 Elsevier Ltd. 保留所有权利。
Even though DNA alkylating agents have been used for many decades in the treatment of cancer, it remains unclear what happens when replication forks encounter alkylated DNA. Here, we used the DNA fibre assay to study the impact of alkylating agents on replication fork progression. We found that the alkylator methyl methanesulfonate (MMS) inhibits replication elongation in a manner that is dose dependent and related to the overall alkylation grade. Replication forks seem to be completely blocked as no nucleotide incorporation can be detected following 1 h of MMS treatment. A high dose of 5 mM caffeine, inhibiting most DNA damage signalling, decreases replication rates overall but does not reverse MMS-induced replication inhibition, showing that the replication block is independent of DNA damage signalling. Furthermore, the block of replication fork progression does not correlate with the level of DNA single-strand breaks. Overexpression of O-6-methylguanine (O6meG)-DNA methyltransferase protein, responsible for removing the most toxic alkylation, O6meG, did not affect replication elongation following exposure to N-methyl-M-nitro-N-nitrosoguanidine. This demonstrates that O6meG lesions are efficiently bypassed in mammalian cells. In addition, we find that MMS-induced gamma H2AX foci co-localise with 53BP1 foci and newly replicated areas, suggesting that DNA double-strand breaks are formed at MMS-blocked replication forks. Altogether, our data suggest that N-alkylations formed during exposure to alkylating agents physically block replication fork elongation in mammalian cells, causing formation of replication-associated DNA lesions, likely double-strand breaks. (C) 2010 Elsevier Ltd. All rights reserved.