Yeast condensin acts as a transient intermolecular crosslinker in entangled DNA

Abstract

Structural-Maintenance-of-Chromosome (SMC) complexes such as condensins organise the folding of chromosomes. However, their role in modulating the entanglement of DNA and chromatin is not fully understood. To address this question, we perform single molecule and bulk characterisation of yeast condensin in entangled DNA. First, we discover that yeast condensin can proficiently bind double-stranded DNA through its hinge domain, in addition to its heads. Through bulk microrheology assays we then discover that physiological concentrations of yeast condensin increase both the viscosity and elasticity of dense solutions of lambda-DNA suggesting that condensin acts as a crosslinker in entangled DNA, stabilising entanglements rather than resolving them and contrasting the popular theoretical picture where SMCs purely drive the formation of segregated, bottle-brush-like chromosome structures. We further discover that the presence of ATP fluidifies the solution -- likely by activating loop extrusion -- but does not recover the viscosity measured in the absence of protein. Finally, we show that the observed rheology can be understood by modelling SMCs as transient crosslinkers in bottle-brush-like entangled polymers. Our findings help us to understand how SMCs affect the dynamics and entanglement of genomes.

Figures (4)

• Figure 1: Yeast condensin proficiently binds dsDNA through its hinge domain.a. Cartoon structure of yeast condensin; different domains are highlighted. b. AlphaFold3 model of structural interaction between the hinge domain and a segment of dsDNA with a small ssDNA bubble in the middle. c. EMSA showing significant binding of the hinge domain (SMC2:K841-L698, SMC4:Q646-F865) to a 25 bp dsDNA oligo in vitro with an estimated $k_D \simeq 0.075-0.15$$\mu$M. d. Fluorescence polarisation assay done with the hinge domain mixed with fluorescently-labelled 50 bp dsDNA oligo and yielding $k_D = 0.7$$\mu$M. e-f. Representative AFM topographs of (e) head-bound and (f) hinge-bound condensin-DNA complexes. Green and lilac arrowheads indicate hinges and core+anchor domains, respectively. g. Quantification of relative hinge and heads bound complexes. Error bars reflect counting statistics $\sqrt{N_i}/N_{total}$. $N_{total} = 299$. h-i. Representative AFM topographs of (h) intra-molecular and (i) inter-molecular condensin-DNA complexes.
• Figure 2: Microrheology reveals that SMCs can form intermolecular bridges in entangled DNA.a-b Sketches of our two hypotheses: a. if condensin performed loop extrusion (intramolecular contacts only), we would expect a solution of entangled linear DNA to be converted into one made of bottle-brush-like polymers, reducing both entanglement and viscoelasticity. b. If condensin performed DNA-bridging (intermolecular contacts), we would expect transient crosslinks. c. The sample made of $\lambda$DNA, condensin, reaction buffer and passive tracers is mixed, incubated and then pipetted in a closed chamber. d. Snapshot of the field of view showing the tracers and short example trajectories (scale bar 20 $\mu$m). e. Mean squared displacement (MSD) of the tracer beads for wild type yeast condensin (WT) in presence and absence of ATP and for a catalytically dead (Q) mutant. For all samples in this figure, DNA concentration is $250$ ng/$\mu$L (or 7.8 nM of $\lambda$DNA) and protein concentration is $0.2$$\mu$M, i.e. about 25 SMCs per DNA molecule. f-g. Elastic ($G^\prime$, f) and viscous ($G^{\prime \prime}$, g) complex moduli obtained from the MSDs through the generalised Stokes Einstein relation. h. Zero-shear viscosity, obtained from the long time behaviour of the MSD. P-values in the plot: $* < 0.05$, $** < 0.01$, $*** < 0.001$. The p-value between WT and Q mutant is 0.13, and hence non significant. i. Elasticity $G^\prime_p$ obtained from the elastic modulus measured at 100 Hz. j. Relaxation time $\tau_R$, obtained as the inverse of the smallest frequency at which $G^\prime$ and $G^{\prime \prime}$ intersect.
• Figure 3: MD simulations of sticky SMCs model the thickening.a. Sketch of DNA with loops formed by SMCs. b. Bead-spring polymer modelling of the region in the dashed box showing the correspondence between patches (cyan) and hinge domain. c. Snapshot from simulations, highlighting intramolecular and intermolecular interactions stabilised by the patches. d Average number of contacts as a function of the number of patches on the beads. e-f. Snapshots of the simulation box in two cases: (e) in equilibrium with no SMC and (f) after loading 50 sticky SMCs per polymer and allowing them to extrude loops. g. Average Mean Squared Displacement (MSD) of the polymers' center of mass (standard deviation shaded) for the control case ($\lambda$-DNA) compared with the cases with SMC but no extrusion (WT) and the case with SMC allowed to extrude loops (WT+ATP). h. Viscosity computed from the stress-relaxation function (see SI) for the three cases in g. Notice that with ATP, the system is more fluid, in line with experiments.
• Figure 4: SMCs act as transient intermolecular crosslinkers that can perform intramolecular loop extrusion in presence of ATP. (top) SMCs loaded on DNA form transient intermolecular bridges by simultaneously binding dsDNA molecules through their heads and hinge domains. (bottom) ATP-driven loop extrusion competes with intermolecular bridging and speeds up DNA dynamics.