As the Watson-Crick faces of nucleobases are protected in double-stranded DNA (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 single-stranded DNA (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 andlays 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面在双链DNA（dsDNA）中受到保护，因此通常假定当DNA在复制和转录过程中变为单链时，对核碱基的Watson-Crick面的有害烷基化损害主要发生。然而，在体外dsDNA中已经报道了对核碱基的Watson-Crick面的破坏。通过不了解的机制。另外，相对于单链DNA（ssDNA），dsDNA所赋予的保护免受甲基化损害的程度尚未确定。dsDNA中的Watson-Crick碱基对与Hoogsteen碱基对处于动态平衡状态，这使嘌呤核碱基的Watson-Crick面暴露于溶剂中。这是否会影响dsDNA的损伤敏感性尚不清楚。使用斑点印迹和引物延伸测定，我们在AT Watson-Crick与Hoogsteen构象中测量了腺嘌呤-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碱基对仅能提供约130倍的腺嘌呤-N1甲基化保护，而对于A（syn）-T Hoogsteen碱基对嵌入DNA-药物复合物中。我们的结果表明，典型dsDNA中的烷基化剂可接近核碱基的Watson-Crick面，而Hoogsteen碱基对可增加这种可接近​​性。鉴于dsDNA相对于ssDNA具有更高的丰度，这些结果表明dsDNA可能是细胞毒性损伤的重要来源。这项工作建立了DMS探测作为一种在体外表征A（syn）-T Hoogsteen碱基对的方法，并为在体内全基因组范围内映射A（syn）-T Hoogsteen和未配对的腺嘌呤的测序方法奠定了基础。