Using DNA flow-stretching assay as a tool to validate the tagging of DNA-binding proteins for single-molecule experiments.

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 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, 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 functio

SUBMITTER: Molina M 

PROVIDER: S-EPMC10055205 | biostudies-literature | 2023 May

REPOSITORIES: biostudies-literature