As the Watson–Crick faces of nucleobases are protected in dsDNA, it is commonly assumed that deleterious alkylation damage to the Watson–Crick faces of nucleobases predominantly occurs when DNA becomes single-stranded during replication and transcription. However, damage to the Watson–Crick faces of nucleobases has been reported in dsDNA in vitro through mechanisms that are not understood. In addition, the extent of protection from methylation damage conferred by dsDNA relative to ssDNA has not been quantified. Watson–Crick base pairs in dsDNA exist in dynamic equilibrium with Hoogsteen base pairs that expose the Watson–Crick faces of purine nucleobases to solvent. Whether this can influence the damage susceptibility of dsDNA remains unknown. Using dot-blot and primer extension assays, we measured the susceptibility of adenine-N1 to methylation by dimethyl sulfate (DMS) when in an A-T Watson–Crick versus Hoogsteen conformation. Relative to unpaired adenines in a bulge, Watson–Crick A-T base pairs in dsDNA only conferred ∼130-fold protection against adenine-N1 methylation, and this protection was reduced to ∼40-fold for A(syn)-T Hoogsteen base pairs embedded in a DNA-drug complex. Our results indicate that Watson–Crick faces of nucleobases are accessible to alkylating agents in canonical dsDNA and that Hoogsteen base pairs increase this accessibility. Given the higher abundance of dsDNA relative to ssDNA, these results suggest that dsDNA could be a substantial source of cytotoxic damage. The work establishes DMS probing as a method for characterizing A(syn)-T Hoogsteen base pairs in vitro and also lays the foundation for a sequencing approach to map A(syn)-T Hoogsteen and unpaired adenines genome-wide in vivo.

中文翻译：

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Hoogsteen 碱基对增加了双链 DNA 对细胞毒性损伤的敏感性。

由于核碱基的 Watson-Crick 面在 dsDNA 中受到保护，因此通常认为，当 DNA 在复制和转录过程中变成单链时，对核碱基的 Watson-Crick 面的有害烷基化损伤主要发生。然而，据报道，体外dsDNA 中核碱基的Watson-Crick 面受损，其机制尚不清楚。此外，dsDNA 相对于 ssDNA 的甲基化损伤保护程度尚未量化。dsDNA 中的 Watson-Crick 碱基对与 Hoogsteen 碱基对处于动态平衡状态，Hoogsteen 碱基对将嘌呤核碱基的 Watson-Crick 面暴露于溶剂中。这是否会影响 dsDNA 的损伤敏感性仍然未知。使用斑点印迹和引物延伸分析，我们测量了在 AT Watson-Crick 与 Hoogsteen 构象中腺嘌呤-N1 对硫酸二甲酯 (DMS) 甲基化的敏感性。相对于凸起中的未配对腺嘌呤，dsDNA 中的 Watson-Crick AT 碱基对仅提供了约 130 倍的腺嘌呤-N1 甲基化保护，对于嵌入的 A(syn)-T Hoogsteen 碱基对，这种保护降低到约 40 倍在 DNA 药物复合物中。我们的结果表明，经典 dsDNA 中的烷化剂可以访问核碱基的 Watson-Crick 面，并且 Hoogsteen 碱基对增加了这种可访问性。鉴于 dsDNA 相对于 ssDNA 的丰度更高，这些结果表明 dsDNA 可能是细胞毒性损伤的重要来源。