Structural maintenance of chromosomes (SMC) complexes organize the genome via DNA loop extrusion. Although some SMCs were reported to do so symmetrically, reeling DNA from both sides into the extruded DNA loop simultaneously, others perform loop extrusion asymmetrically toward one direction only. The mechanism underlying this variability remains unclear. Here, we examine the directionality of DNA loop extrusion by SMCs using in vitro single-molecule experiments. We find that cohesin and SMC5/6 do not reel in DNA from both sides, as reported before, but instead extrude DNA asymmetrically, although the direction can switch over time. Asymmetric DNA loop extrusion thus is the shared mechanism across all eukaryotic SMC complexes. For cohesin, direction switches strongly correlate with the turnover of the subunit NIPBL, during which DNA strand switching may occur. Apart from expanding by extrusion, loops frequently diffuse and shrink. The findings reveal that SMCs, surprisingly, can switch directions.

Human cohesin extrudes DNA into loops and is positioned along the genome by stalling at the human CCCTC-binding factor (CTCF) upon encountering its N-terminal region (NTR). The mechanism underlying this stalling, however, is unresolved. Using single-molecule assays that monitor DNA loop extrusion (LE) in the presence of NTR fragments, we identify two amino acid motifs, YDF and KTYQR, which hinder LE. KTYQR is found to completely block LE activity, while YDF hinders cohesin from completing LE step cycles and converts cohesin into a unidirectional extruder by strengthening the affinity of STAG1 to DNA. We thus identify two distinct NTR motifs that stall LE via different yet synergistic mechanisms, highlighting the multifaceted ways employed by CTCF to modulate LE to shape and regulate genomes.

Eukaryotes carry three types of structural maintenance of chromosome (SMC) protein complexes, condensin, cohesin, and SMC5/6, which are ATP-dependent motor proteins that remodel the genome via DNA loop extrusion (LE). SMCs modulate DNA supercoiling but remains incompletely understood how this is achieved. Using a single-molecule magnetic tweezers assay that directly measures how much twist is induced by individual SMCs in each LE step, we demonstrate that all three SMC complexes induce the same large negative twist (i.e., linking number change ΔL _k of ~−0.6 at each LE step) into the extruded loop, independent of step size and DNA tension. Using ATP hydrolysis mutants and nonhydrolyzable ATP analogs, we find that ATP binding is the twist-inducing event during the ATPase cycle, coinciding with the force-generating LE step. The fact that all three eukaryotic SMC proteins induce the same amount of twist indicates a common DNA-LE mechanism among these SMC complexes.

Correction to: Scientific Reports, published online 19 May 2023 The original version of this Article contained an error in Figure 1b-1, where the fore- and background order of the strands “DNA” (in black) and “Brn1 Kleisin” (in green), were switched. The original Figure 1 and accompanying legend appear below. (Figure presented.) Description of the mechanism postulated by Shaltiel et al. for roadblock passage into an extruded loop on the DNA and a potential nontopological model. (a) The steps through the proposed DNA loop extrusion cycle are commented in more detail in steps 1–6 within the figure. Adapted from Ref.¹¹. (b) Potential nontopological model which is closely analogous to the pseudotopological model, but with a slight variation in the DNA-SMC topology which allows particle bypass. The original Article has been corrected.

A potential mechanism of DNA loop extrusion by molecular motors is discussed.

In eukaryotes, genomic DNA is extruded into loops by cohesin¹. By restraining this process, the DNA-binding protein CCCTC-binding factor (CTCF) generates topologically associating domains (TADs)^2,3 that have important roles in gene regulation and recombination during development and disease^1,4–7. How CTCF establishes TAD boundaries and to what extent these are permeable to cohesin is unclear⁸. Here, to address these questions, we visualize interactions of single CTCF and cohesin molecules on DNA in vitro. We show that CTCF is sufficient to block diffusing cohesin, possibly reflecting how cohesive cohesin accumulates at TAD boundaries, and is also sufficient to block loop-extruding cohesin, reflecting how CTCF establishes TAD boundaries. CTCF functions asymmetrically, as predicted; however, CTCF is dependent on DNA tension. Moreover, CTCF regulates cohesin’s loop-extrusion activity by changing its direction and by inducing loop shrinkage. Our data indicate that CTCF is not, as previously assumed, simply a barrier to cohesin-mediated loop extrusion but is an active regulator of this process, whereby the permeability of TAD boundaries can be modulated by DNA tension. These results reveal mechanistic principles of how CTCF controls loop extrusion and genome architecture.

DNA loop extrusion by structural-maintenance-of-chromosome (SMC) complexes has emerged as a primary organizing principle for chromosomes. The mechanism by which SMC motor proteins extrude DNA loops is still unresolved and much debated. The ring-like structure of SMC complexes prompted multiple models where the extruded DNA is topologically or pseudotopologically entrapped within the ring during loop extrusion. However, recent experiments showed the passage of roadblocks much bigger than the SMC ring size, suggesting a nontopological mechanism. Recently, attempts were made to reconcile the observed passage of large roadblocks with a pseudotopological mechanism. Here we examine the predictions of these pseudotopological models and find that they are not consistent with new experimental data on SMC roadblock encounters. Particularly, these models predict the formation of two loops and that roadblocks will reside near the stem of the loop upon encounter—both in contrast to experimental observations. Overall, the experimental data reinforce the notion of a nontopological mechanism for extrusion of DNA.

Ring-shaped structural maintenance of chromosomes (SMC) complexes like condensin and cohesin extrude loops of DNA. It remains, however, unclear how they can extrude DNA loops in chromatin that is bound with proteins. Here, we use in vitro single-molecule visualization to show that nucleosomes, RNA polymerase, and dCas9 pose virtually no barrier to loop extrusion by yeast condensin. We find that even DNA-bound nanoparticles as large as 200 nm, much bigger than the SMC ring size, also translocate into DNA loops during extrusion by condensin and cohesin. This even occurs for a single-chain version of cohesin in which the ring-forming subunits are covalently linked and cannot open to entrap DNA. The data show that SMC-driven loop extrusion has surprisingly little difficulty in accommodating large roadblocks into the loop. The findings also show that the extruded DNA does not pass through the SMC ring (pseudo)topologically, hence pointing to a nontopological mechanism for DNA loop extrusion.