A single molecule TIRF-based platform to study DNA supercoiling effect on DNA processing enzymes.

Abstract

DNA topology plays an important role in regulating DNA metabolic pathways. DNA supercoiling changes the DNA properties and proteins may differentially interact with DNA under various supercoiling states. A biophysical investigation of how proteins differentiate various topological states could further our understanding of protein-DNA interactions. We developed new single molecule total internal reflection fluorescence (smTIRF) -based platform to investigate how DNA topology affects protein-DNA interactions. We used this platform to study MutS, which detects a mismatched base pair to initiate mismatch repair, and CRISPR-Cas9. We generated plasmids that are site-specifically labeled with one or two fluorophores and a biotin. We prepared them in three different topological states: “Linear”, “relaxed circular”, and “negatively supercoiled”. For MutS study, we designed a Cy5 labeled plasmid with an adjacent mismatch. We observed FRET between Cy3-MutS and Cy5 when MutS bound the mismatch. In the presence of ADP, MutS bound the negative supercoiled DNA with a longer residence time compared to the relaxed circular DNA, suggesting that negative supercoiling helps mismatch recognition. For CRISPR-Cas9, we created a dual labeled plasmid to observe R-loop formation induced by dCas9. R-loop formation rate was similar for all three DNA topological states when the guide RNA sequence matched the target DNA sequence, while the rate was highest for the negatively supercoiled DNA and decreased progressively for the relaxed circular DNA and the linear DNA when we introduced one mismatch in either the PAM (protospacer adjacent motif)-proximal position or the PAM-distal position. Our observation confirms that DNA negative supercoiling assists DNA unwinding by Cas9, and this can exacerbate off-target effects in the cell. Collectively, our smTIRF-based platform allows high throughput investigation of supercoiling effects on DNA protein interactions as demonstrated using two different biological systems.