Molecular Crowding Suppresses Mechanical Stress-Driven DNA Strand Separation

ABSTRACT Molecular crowding influences DNA mechanics and DNA - protein interactions and is ubiquitous in living cells. Quantifying the effects of molecular crowding on DNA supercoiling is essential to relating in-vitro experiments to in-vivo DNA supercoiling. We use single molecule magnetic tweezers to study DNA supercoiling in the presence of dehydrating or crowding co-solutes. To study DNA supercoiling, we apply a stretching force of 0.8 pN to the DNA and then rotate one end of the DNA to induce supercoiling. In a 200 mM NaCl buffer without co-solutes, negatively supercoiled DNA absorbs some of the tortional stress by forming locally melted DNA regions. The base-pairs in these locally melted regions are believed to adopt a configuration where nucleotide base pairing is disrupted. We find that the presence of a dehydrating co-solute like glycerol further destabilizes base-pairs in negatively supercoiled DNA. The presence of polyethylene glycol, commonly used as a crowding agent, suppresses local strand separation and results in plectoneme formation even when DNA is negatively supercoiled. The results presented in this letter suggest further directions for studies of DNA supercoiling and supercoiled DNA – protein interactions in molecular conditions that approximate in-vivo molecular composition. SIGNIFICANCE Accurate modelling of DNA mechanics is central to interpreting results of single molecule studies of DNA mechanics and DNA-protein interactions. While the effect of molecular conditions on thermal stability of short and relaxed DNA has been studied, the influence of molecular conditions on DNA supercoiling has not been explored. We present the first single molecule study of DNA supercoiling in the presence of crowding and dehydrating co-solutes. We observe that co-solutes can increase or completely suppress mechanical stress-driven base-pair disruption in negatively supercoiled DNA. This change of DNA supercoiling is likely to significantly affect the function of DNA-binding proteins. Our results motivate the need for systematic exploration of DNA supercoiling in presence of co-solutes to accurately relate in-vitro DNA-protein interactions to in-vivo DNA-protein interactions.