Precise mapping of single-stranded DNA breaks by sequence-templated erroneous DNA polymerase end-labelling

Leonie Wenson, Johan Heldin, Marcel Martin, Yücel Erbilgin, Barış Salman, Anders Sundqvist, Wesley Schaal, Friederike A. Sandbaumhüter, Erik T. Jansson, Xingqi Chen, Anton Davidsson, Bo Stenerlöw, Jaime A. Espinoza, Mikael Lindström, Johan Lennartsson, Ola Spjuth and Ola Söderberg ()
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Leonie Wenson: Uppsala University, Biomedical Center
Johan Heldin: Uppsala University, Biomedical Center
Marcel Martin: Stockholm University
Yücel Erbilgin: Istanbul University
Barış Salman: Istanbul University
Anders Sundqvist: Uppsala University, Biomedical Center
Wesley Schaal: Uppsala University, Biomedical Center
Friederike A. Sandbaumhüter: Uppsala University, Biomedical Center
Erik T. Jansson: Uppsala University, Biomedical Center
Xingqi Chen: Uppsala University, Biomedical Center
Anton Davidsson: Uppsala University, Biomedical Center
Bo Stenerlöw: Uppsala University, Biomedical Center
Jaime A. Espinoza: Uppsala University, Biomedical Center
Mikael Lindström: Karolinska Institutet
Johan Lennartsson: Uppsala University, Biomedical Center
Ola Spjuth: Uppsala University, Biomedical Center
Ola Söderberg: Uppsala University, Biomedical Center

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract 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 × 10−6 with an enrichment in active promoter regions.

Date: 2025
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DOI: 10.1038/s41467-025-62512-4

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