Type IA topoisomerases cleave single-stranded DNA and relieve negative supercoils in discrete steps corresponding to the passage of the intact DNA strand through the cleaved strand. Although it is assumed type IA topoisomerases accomplish this strand passage via a protein-mediated DNA gate, opening of this gate has never been observed. We developed a single-molecule assay to directly measure gate opening of the E. coli type IA topoisomerases I and III. We found that following cleavage of single-stranded DNA, the protein gate opens by as much as 6.6 nm and can close against forces in excess of 16 pN. Key differences in the cleavage, ligation and gate dynamics of these two enzymes provide insights into their different cellular functions. The single-molecule results are broadly consistent with conformational changes obtained from molecular dynamics simulations. These results allow us to develop a mechanistic model of type IA topoisomerase-ssDNA interactions.

During microRNA (miRNA) biogenesis, the Microprocessor complex (MC), composed minimally of Drosha, an RNaseIII enzyme, and DGCR8, a double-stranded RNA-binding protein, cleaves the primary-miRNA (pri-miRNA) to release the pre-miRNA stemloop structure. Size-exclusion chromatography of the MC, isolated from mammalian cells, suggested multiple copies of one or both proteins in the complex. However, the exact stoichiometry was unknown. Initial experiments suggested that DGCR8 bound pri-miRNA substrates specifically, and given that Drosha could not be bound or cross-linked to RNA, a sequential model for binding was established in which DGCR8 bound first and recruited Drosha. Therefore, many laboratories have studied DGCR8 binding to RNA in the absence of Drosha and have shown that deletion constructs of DGCR8 can multimerize in the presence of RNA. More recently, it was demonstrated that Drosha can bind pri-miRNA substrates in the absence of DGCR8, casting doubt on the sequential model of binding. In the same study, using a single-molecule photobleaching assay, fluorescent protein-tagged deletion constructs of DGCR8 and Drosha assembled into a heterotrimeric complex on RNA, comprising two DGCR8 molecules and one Drosha molecule. To determine the stoichiometry of Drosha and DGCR8 within the MC in the absence of added RNA, we also used a single-molecule photobleaching assay and confirmed the heterotrimeric model of the human MC. We demonstrate that a heterotrimeric complex is likely preformed in the absence of RNA and exists even when full-length proteins are expressed and purified from human cells, and when hAGT-derived tags are used rather than fluorescent proteins.

The mechanism of DNA compaction by dendrimers is key to the design of nanotechnologies that can deliver genetic material into cells. We present atomistic simulations, mesoscopic modeling and single-molecule pulling experiments describing DNA dendrimer interactions. All-atom molecular dynamics were used to characterize pulling-force-dependent interactions between DNA and generation-3 PAMAM amine-terminated dendrimers, and a free energy profile and mean forces along the interaction coordinate are calculated. The energy, force, and geometry parameters computed at the atomic level are input for a Monte Carlo model yielding mesoscopic force-extension curves. Actual experimental single-molecule curves obtained with optical tweezers are also presented, and they show remarkable agreement with the virtual curves from our model. The calculations reveal the microscopic origin of the hysteresis observed in the phase transition underlying compaction. A broad range of ionic and pulling parameters is sampled, and suggestions for windows of conditions to probe new single-molecule behavior are made.