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Precise mapping of single-stranded DNA breaks by using an engineered, error-prone DNA polymerase for sequence-templated erroneous end-labelling
Open accessPreprint — published as 10.1038/s41467-025-62512-4 · Paper page
Abstract
The ability to analyze whether DNA includes lesions is important in identifying mitogenic substances. Until now, the detection of single-stranded DNA breaks (SSBs) has lacked precise methods. To overcome this limitation, we have engineered a chimeric DNA polymerase, Sloppymerase, that is able to replicate DNA in the absence of one nucleotide. In addition to polymerase activity, Sloppymerase demonstrates 5´-3´exonuclease activity. We characterized the activity of Sloppymerase and utilized the enzyme to develop a method for sequence-templated erroneous end-labelling sequencing (STEEL-seq) that is relevant to the mapping of SSBs. Following the omission of a specific nucleotide, e.g., dATP, from the reaction mixture, Sloppymerase introduces mismatches directly downstream of SSBs at positions that should contain deoxyadenosine. The ability to retain sequence information after end-labelling ensures that hits are bona fide SSBs. STEEL-seq works with a variety of sequencing technologies, shown by our successful experiments using Sanger, Illumina, PacBio and Nanopore systems.
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Metadata
- Authors
- Söderberg, O., Wenson, L., Heldin, J., Martin, M., Erbilgin, Y., Salman, B., Schaal, W., Sandbaumhüter, F., Jansson, E., Chen, X., Davidsson, A., Stenerlöw, B., & Spjuth, O.
- Affiliations
- Uppsala University (SE)
- Stockholm University (SE)
- Istanbul University (TR)
- Corresponding author
- Söderberg, Ola
- ORCID
- Venue
- Nature Communications
- Published
- 2024-10-07
- Volume
- 16
- Issue
- 1
- Pages
- 7130-7130
- ISSN
- 2041-1723 (ISSN-L)
- PMC
- PMC12322144
- OpenAlex
- W4412947224
- Cited by
- 1
- References
- 1
- Citations by year
- 2026: 1
- FWCI
- 0.30
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Nature Communications (publishedVersion, cc-by)
- Research Square (acceptedVersion, cc-by)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- Diva portal (Dalarna University Library) (submittedVersion, other-oa)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
- Funders
- Swedish Cancer Foundation (22 2306 Pj)
- Cancerfonden (222306Pj)
- Knut och Alice Wallenbergs Stiftelse (KAW 2020.0239, 2017.0003, KAW 2017.0003, 2020.0239)
- Vetenskapsrådet
- Uppsala Universitet
- Science for Life Laboratory (KAW 2020.0239)
- Uppsala Multidisciplinary Center for Advanced Computational Science
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › DNA Repair Mechanisms
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › Carcinogens and Genotoxicity Assessment
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Phylogenetic Studies
- Keywords
- single-strand breaks, error-prone DNA polymerases, mutagen detection, promoter
- MeSH terms
- DNA-Directed DNA Polymerase, DNA Replication, DNA, Single-Stranded, Humans, Nucleic Acid Conformation, Protein Engineering, Sequence Analysis, DNA, DNA Breaks, Single-Stranded, High-Throughput Nucleotide Sequencing
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