A high throughput single molecule platform to study DNA supercoiling effect on protein-DNA interactions.

ABSTRACT: DNA supercoiling significantly influences DNA metabolic pathways. To examine its impact on DNA-protein interactions at the single-molecule level, we developed a highly efficient and reliable protocol to modify plasmid DNA at specific sites, allowing us to label plasmids with fluorophores and biotin. We then induced negative and positive supercoiling in these plasmids using gyrase and reverse gyrase, respectively. Comparing supercoiled DNA with relaxed circular DNA, we assessed the effects of supercoiling on CRISPR-Cas9 and mismatch repair protein MutS. We found that negative DNA supercoiling exacerbates off-target effects in DNA unwinding by Cas9. For MutS, we observed both negative and positive DNA supercoiling enhances the binding interaction between MutS and a mismatched base pair but d

SUBMITTER: Lee H 

PROVIDER: S-EPMC11527113 | biostudies-literature | 2024 Oct

REPOSITORIES: biostudies-literature