Measuring bridging forces in protein-DNA condensates

Abstract

Protein-DNA condensates mediate transcription and regulate gene expression and DNA replication and repair. The intermolecular bridging forces stabilizing condensates have direct roles in these processes. Here we use optical tweezers to measure bridging forces. In the presence of protamine, a single condensate is observed on a 20.5-knt single-stranded DNA (ssDNA) tethered between two microbeads. Stretching produces force curves with a sawtooth pattern, suggesting that the condensate is dissembled by the sequential rupture of individual protamine-ssDNA bridges. The bridging forces are 11.3 {+/-} 4.6 pN, with unfolding lengths of 1.3 {+/-} 0.8 {micro}m for single bridges. In contrast, double-stranded DNA (dsDNA) forms protamine-bridged tangles that can withstand forces high enough ([~]55 pN) for strand separation. ssDNA tracks unpeeled at nicks on dsDNA by overstretching seed tangle formation upon retraction, but the initial condensates have a sufficient ssDNA-to-dsDNA ratio to appear liquid-like, as indicated by a sawtooth pattern in the subsequent stretching. The presence of dsDNA raises bridging forces to 34 {+/-} 8 pN, which revert to [~]10 pN upon adding external ssDNA. In line with these single-molecule results, protamine-dsDNA mixtures form solid-like aggregates and require the addition of ssDNA to become liquid droplets. Conversely, adding dsDNA slows the fusion of protamine-ssDNA droplets. This work demonstrates the first measurements of bridging forces and shows that the ssDNA-to-dsDNA ratio can tune their magnitude in protein-DNA condensates.