Wenson Leonie, Heldin Johan, Martin Marcel, Erbilgin Yücel, Salman Barış, Sundqvist Anders, Schaal Wesley, Sandbaumhüter Friederike A, Jansson Erik T, Chen Xingqi, Davidsson Anton, Stenerlöw Bo, Espinoza Jaime A, Lindström Mikael, Lennartsson Johan, Spjuth Ola, Söderberg Ola
Department of Pharmaceutical Biosciences, Science for Life Laboratory, Uppsala University, Biomedical Center, Uppsala, Sweden.
Department of Biochemistry and Biophysics, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Stockholm University, Solna, Sweden.
Nat Commun. 2025 Aug 4;16(1):7130. doi: 10.1038/s41467-025-62512-4.
The ability to analyze whether DNA contains lesions is essential in identifying mutagenic substances. Currently, the detection of single-stranded DNA breaks (SSBs) lacks precision. To address this limitation, we develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq), which enables the mapping of SSBs. The method requires a highly error-prone DNA polymerase, so we engineer a chimeric DNA polymerase, Sloppymerase, capable of replicating DNA in the absence of one nucleotide. Following the omission of a specific nucleotide (e.g., dATP) from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions where deoxyadenosine should occur. This mismatch pattern, coupled with the retention of sequence information flanking these sites, ensures that the identified hits are bona fide SSBs. STEEL-seq is compatible with a variety of sequencing technologies, as demonstrated using Sanger, Illumina, PacBio, and Nanopore systems. Using STEEL-seq, we determine the SSB/base pair frequency in the human genome to range between 0.7 and 3.8 × 10 with an enrichment in active promoter regions.
分析DNA是否含有损伤的能力对于识别诱变物质至关重要。目前，单链DNA断裂（SSB）的检测缺乏精确性。为了解决这一局限性，我们开发了一种序列模板错误末端标记测序方法（STEEL-seq），该方法能够对SSB进行定位。该方法需要一种高度易错的DNA聚合酶，因此我们设计了一种嵌合DNA聚合酶——草率聚合酶，它能够在缺少一种核苷酸的情况下复制DNA。在从反应混合物中省略特定核苷酸（例如dATP）后，草率聚合酶会在SSB下游紧邻脱氧腺苷应出现的位置引入错配。这种错配模式，再加上这些位点侧翼序列信息的保留，确保了所识别的命中位点是真正的SSB。如使用桑格、Illumina、PacBio和纳米孔系统所证明的，STEEL-seq与多种测序技术兼容。使用STEEL-seq，我们确定人类基因组中SSB/碱基对频率在0.7至3.8×10之间，且在活跃启动子区域有所富集。
