To establish relationships between mutations, for example in cancer genomes, and possible mechanisms linked to DNA damage, it is necessary to know at what sequence positions of the genome the damage occurs. However, it has been challenging to specifically map DNA damage at the nucleotide level of resolution and genome-wide with high sensitivity. Here, we describe a new method, which we named circle damage sequencing (circle-damage-seq), to accomplish this goal. The method is based on circularization of DNA molecules and DNA damage-selective cleavage of the circularized DNA followed by adapter ligation and sequencing. Based on the design of this approach, only DNA damage-containing molecules are sequenced. We conducted proof-of-principle studies to show that mapping of ultraviolet B-induced cyclobutane pyrimidine dimers (CPDs) can easily be achieved and show a specific tetranucleotide sequence context for CPDs (PyPy-PyT/A) with no further sequence enrichment outside of this context. Our approach shows strongly reduced levels of CPDs near transcription start sites and a spike of this damage near the transcription end sites of genes. We then show that 1,N6-etheno-deoxyadenosine DNA adducts formed after treatment of cells with the lipid peroxidation product 4-hydroxynonenal can be mapped genome-wide at adenine positions within a preferred sequence context of 5TAC/G3. The circle-damage-seq method can be adapted for a variety of DNA lesions for which specific excision enzymes are available.

中文翻译：

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通过圆环损伤测序以核苷酸分辨率绘制全基因组DNA损伤图

为了建立例如癌症基因组中的突变与与DNA损伤相关的可能机制之间的关系，有必要知道在基因组的什么序列位置发生损伤。然而，挑战性的是以高灵敏度灵敏地在分辨率的核苷酸水平和全基因组范围内特异性定位DNA损伤。在这里，我们描述了一种新的方法，我们将其称为圆损伤定序（circle-damage-seq），以实现这一目标。该方法基于DNA分子的环化和环化DNA的DNA损伤选择性切割，然后进行衔接子连接和测序。基于这种方法的设计，仅对包含DNA损伤的分子进行测序。我们进行了原理验证研究，以表明可以轻松实现紫外线B诱导的环丁烷嘧啶二聚体（CPD）的作图，并显示了CPD的特定四核苷酸序列背景（PyPy-PyT / A），在序列外没有其他序列富集在这种情况下。我们的方法显示，在转录起始位点附近的CPD含量大大降低，并且在基因的转录终止位点附近这种破坏的峰值。然后，我们表明，在用脂质过氧化产物4-hydroxynonenal处理细胞后形成的1，N6-乙氧基-脱氧腺苷DNA加合物可以在5TAC / G3的优选序列范围内在腺嘌呤位置定位全基因组范围。圆周损伤seq方法可适用于多种DNA损伤，可使用这些酶进行特异性切除。