Condensin is a conserved SMC complex that uses its ATPase machinery to structure genomes, but how it does so is largely unknown. We show that condensin's ATPase has a dual role in chromosome condensation. Mutation of one ATPase site impairs condensation, while mutating the second site results in hyperactive condensin that compacts DNA faster than wild-type, both in vivo and in vitro. Whereas one site drives loop formation, the second site is involved in the formation of more stable higher-order Z loop structures. Using hyperactive condensin I, we reveal that condensin II is not intrinsically needed for the shortening of mitotic chromosomes. Condensin II rather is required for a straight chromosomal axis and enables faithful chromosome segregation by counteracting the formation of ultrafine DNA bridges. SMC complexes with distinct roles for each ATPase site likely reflect a universal principle that enables these molecular machines to intricately control chromosome architecture.

The nuclear pore complex (NPC) is the gatekeeper for nuclear transport in eukaryotic cells. A key component of the NPC is the central shaft lined with intrinsically disordered proteins (IDPs) known as FG-Nups, which control the selective molecular traffic. Here, we present an approach to realize artificial NPC mimics that allows controlling the type and copy number of FG-Nups. We constructed 34 nm-wide 3D DNA origami rings and attached different numbers of NSP1, a model yeast FG-Nup, or NSP1-S, a hydrophilic mutant. Using (cryo) electron microscopy, we find that NSP1 forms denser cohesive networks inside the ring compared to NSP1-S. Consistent with this, the measured ionic conductance is lower for NSP1 than for NSP1-S. Molecular dynamics simulations reveal spatially varying protein densities and conductances in good agreement with the experiments. Our technique provides an experimental platform for deciphering the collective behavior of IDPs with full control of their type and position.

It has been hypothesized that SMC protein complexes such as condensin and cohesin spatially organize chromosomes by extruding DNA into large loops. We directly visualized the formation and processive extension of DNA loops by yeast condensin in real time. Our findings constitute unambiguous evidence for loop extrusion. We observed that a single condensin complex is able to extrude tens of kilobase pairs of DNA at a force-dependent speed of up to 1500 base pairs per second, using the energy of adenosine triphosphate hydrolysis. Condensin-induced loop extrusion was strictly asymmetric, which demonstrates that condensin anchors onto DNA and reels it in from only one side. Active DNA loop extrusion by SMC complexes may provide the universal unifying principle for genome organization.

The three-dimensional organization of DNA is increasingly understood to play a decisive role in vital cellular processes. Many studies focus on the role of DNA-packaging proteins, crowding, and confinement in arranging chromatin, but structural information might also be directly encoded in bare DNA itself. Here, we visualize plectonemes (extended intertwined DNA structures formed upon supercoiling) on individual DNA molecules. Remarkably, our experiments show that the DNA sequence directly encodes the structure of supercoiled DNA by pinning plectonemes at specific sequences. We develop a physical model that predicts that sequence-dependent intrinsic curvature is the key determinant of pinning strength and demonstrate this simple model provides very good agreement with the data. Analysis of several prokaryotic genomes indicates that plectonemes localize directly upstream of promoters, which we experimentally confirm for selected promotor sequences. Our findings reveal a hidden code in the genome that helps to spatially organize the chromosomal DNA.

Finding the target site and associating in a specific orientation are essential tasks for DNA-binding proteins. In order to make the target search process as efficient as possible, proteins should not only rapidly diffuse to the target site but also dynamically explore multiple local configurations before diffusing away. Protein flipping is an example of this second process that has been observed previously, but the underlying mechanism of flipping remains unclear. Here, we probed the mechanism of protein flipping at the single molecule level, using HIV-1 reverse transcriptase (RT) as a model system. In order to test the effects of long-range attractive forces on flipping efficiency, we varied the salt concentration and macromolecular crowding conditions. As expected, increased salt concentrations weaken the binding of RT to DNA while increased crowding strengthens the binding. Moreover, when we analyzed the flipping kinetics, i.e. the rate and probability of flipping, at each condition we found that flipping was more efficient when RT bound more strongly. Our data are consistent with a view that DNA bound proteins undergo multiple rapid re-binding events, or short hops, that allow the protein to explore other configurations without completely dissociating from the DNA.

DNA supercoiling crucially affects cellular processes such as DNA replication, gene expression, and chromatin organization. However, mechanistic understanding of DNA supercoiling and the related DNA-processing enzymes has remained limited, mainly due to the lack of convenient experimental tools to probe these phenomena. Here, we report a novel high-throughput single-molecule assay for real-time visualization of supercoiled DNA molecules, named ISD (Intercalation-induced Supercoiling of DNA). We use an intercalating dye to induce supercoiling of surface-attached DNA molecules as well as to visualize coiled-loop structures (i.e., plectonemes) formed on DNA. The technique is solely based on epifluorescence microscopy and requires no mechanical manipulation of the DNA molecules. This new assay allows to track positions and sizes of individual plectonemes and characterize their position-dependent dynamics such as nucleation, termination, and diffusion. We describe the ISD technique and demonstrate its potential by establishing that plectonemes are pinned to a local 10-nucleotide long mispaired sequence along a double-stranded DNA molecule.