ArticleCell reports methods2023
A framework to validate fluorescently labeled DNA-binding proteins for single-molecule experiments.
Article in Cell reports methods, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 6 papers.
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Who cites it
6 citing papers in PubMed.
- Bacillus subtilis ParB C-terminal lysine residues are essential for dimerization and in vivo function, indicating their roles in DNA sliding.Nucleic acids research · 2026Article
- Probing the effect of PEG-DNA interactions and buffer viscosity on tethered DNA in shear flow.bioRxiv : the preprint server for biology · 2025Article
- Single-molecule DNA-flow stretching assay as a versatile hybrid tool for investigating DNA-protein interactions.BMB reports · 2025Review
- Probing the effect of PEG-DNA interactions and buffer viscosity on tethered DNA in shear flow.PloS one · 2025Article
- Connecting the dots: key insights on ParB for chromosome segregation from single-molecule studies.FEMS microbiology reviews · 2024Review
- Mind your tag in single-molecule measurements.Cell reports methods · 2023Article
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8 authors.
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Abstract
Due to the enhanced labeling capability of maleimide-based fluorescent probes, lysine-cysteine-lysine (KCK) tags are frequently added to proteins for visualization. In this study, we employed an in vitro single-molecule DNA flow-stretching assay as a sensitive way to assess the impact of the KCK tag on the property of DNA-binding proteins. Using Bacillus subtilis ParB as an example, we show that, although no noticeable changes were detected by in vivo fluorescence imaging and chromatin immunoprecipitation (ChIP) assays, the KCK tag substantially altered ParB's DNA compaction rates and its response to nucleotide binding and to the presence of the specific sequence (parS) on the DNA. While it is typically assumed that short peptide tags minimally perturb protein function, our results urge researchers to carefully validate the use of tags for protein labeling. Our comprehensive analysis can be expanded and used as a guide to assess the impacts of other tags on DNA-binding proteins in single-molecule assays.
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