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. 2017 Nov 3;358(6363):672-676.
doi: 10.1126/science.aan6516. Epub 2017 Sep 7.

The Condensin Complex Is a Mechanochemical Motor That Translocates Along DNA

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The Condensin Complex Is a Mechanochemical Motor That Translocates Along DNA

Tsuyoshi Terakawa et al. Science. .
Free PMC article


Condensin plays crucial roles in chromosome organization and compaction, but the mechanistic basis for its functions remains obscure. We used single-molecule imaging to demonstrate that Saccharomyces cerevisiae condensin is a molecular motor capable of adenosine triphosphate hydrolysis-dependent translocation along double-stranded DNA. Condensin's translocation activity is rapid and highly processive, with individual complexes traveling an average distance of ≥10 kilobases at a velocity of ~60 base pairs per second. Our results suggest that condensin may take steps comparable in length to its ~50-nanometer coiled-coil subunits, indicative of a translocation mechanism that is distinct from any reported for a DNA motor protein. The finding that condensin is a mechanochemical motor has important implications for understanding the mechanisms of chromosome organization and condensation.


Fig. 1
Fig. 1. Biochemistry of budding yeast condensin holocomplexes
(A) Schematic of the S. cerevisiae condensin complex. The Brn1 kleisin subunit connects the ATPase head domains of the Smc2-Smc4 heterodimer and recruits the HEAT-repeat subunits Ycs4 and Ycg1. The cartoon highlights the position of the HA3 tag used for labeling. (B) Conceptual schematic of loop extrusion for models with either two (top) or one (bottom) DNA strand(s) passing through the center of the SMC ring. (C) Wild-type and ATPase-deficient Smc2(Q147L)-Smc4(Q302L) condensin complexes analyzed by SDS–polyacrylamide gel electrophoresis and Coomassie staining (Q, glutamine; L, leucine). (D) Electron micrographs of wild-type condensin holocomplexes rotary-shadowed with platinum/carbon. Scale bars, 100 nm. (E) Electrophoretic mobility shift assays with a 6-carboxyfluorescein–labeled 45-bp dsDNA substrate (100 nM) and the indicated protein concentrations. (F) ATP hydrolysis by wild-type and ATPase mutant condensin complexes (0.5 μM) upon addition of increasing concentrations of a 6.4-kb linear DNA at saturated ATP concentrations (5 mM). The plot shows means ± SD from three (wild-type) or two (ATPase mutant) independent experiments. (G) Michaelis-Menten kinetics for the rate of ATP hydrolysis by wild-type condensin complexes (0.5 μM) at increasing ATP concentrations in the presence of 240 nM 6.4-kb linear DNA. The plot shows means ± SD from three independent experiments. The fit corresponds to a Km of 0.4 ± 0.07 mM for ATP and a kcat of 2.0 ± 0.1 s−1 per molecule of condensin (mean ± SE).
Fig. 2
Fig. 2. DNA curtain assay for DNA binding activity of condensin
(A) Schematic of the double-tethered DNA curtain assay (up and down arrows, inlet and outlet of buffer, respectively). (B) Still images showing Qdot-tagged condensin (magenta) bound to YoYo1-stained DNA (green). (C) Kymograph showing examples of Qdot-tagged condensin translocating on a single DNA molecule (unlabeled); the initial condensin binding sites, dissociation positions, and collisions with the barriers or pedestals are highlighted with color-coded arrowheads. (D) Kymograph showing Qdot-tagged ATPase-deficient mutant Smc2(Q147L)-Smc4(Q302L) condensin undergoing 1D diffusion on DNA (unlabeled). (E) Initial binding site and (F) dissociation site distributions of condensin superimposed on the A/T content of the λ-DNA substrate. All reactions contained 4 mM ATP. Error bars in (E) and (F) represent SD calculated by boot strap analysis. kbp, kilobase pairs.
Fig. 3
Fig. 3. Condensin is an ATP-dependent mechanochemical molecular motor
(A) Examples of tracked translocation trajectories for Qdot-tagged wild-type condensin and (B) for the ATPase-deficient Smc2(Q147L)-Smc4(Q302L) condensin mutant. (C) Mean squared displacement (MSD) plots for wild-type condensin and (D) for the ATPase-deficient mutant, obtained from the tracked trajectories. The inset in (D) is a magnification of the main curves. (E) Velocity distributions for condensin translocation activity. The dashed line is a log-normal fit to the translocation rate data. (F) Processivity measurements of condensin motor activity. The dashed line highlights the translocation distance corresponding to dissociation of one half of the bound condensin complexes. Error bars represent SD calculated by boot strap analysis.
Fig. 4
Fig. 4. Coupling condensin motor activity to DNA loop extrusion
(A) Minimal mechanistic framework necessary for coupling ATP-dependent translocation to the extrusion of a cis DNA loop. In this generic model, a motor domain (green) must move away from another DNA binding domain (blue), and the latter domain can either remain stationary (as depicted) or act as a motor domain and move in the opposite direction (not depicted). (B) Detection of cis loop extrusion is not possible when the DNA is held in a fixed configuration, as in the double-tethered curtain configuration that allows for direct detection of condensin motor activity in the absence of condensation (top). The middle and bottom panels show a schematic of an assay to mimic cis DNA loop extrusion by providing a second λ-DNA substrate in trans. (C) Examples of kymographs showing translocation of a second λ-DNA substrate (stained with YoYo1) provided in trans in the presence of unlabeled condensin. The presence of the trans DNA substrate is revealed as regions of locally high YoYo1 signal intensity, as highlighted by arrowheads. The regions of higher signal intensity are not detected when the trans DNA is omitted from the reaction. (D) Velocity distribution histogram and (E) survival probability plot for condensin bound to the trans DNA substrate. The dashed line in (E) highlights the translocation distance corresponding to dissociation of one half of the bound condensin complexes. Error bars represent SD calculated by boot strap analysis. Cartoons of generalized models for condensin motor activity through (F) “scrunching” or (G) “walking” mechanisms, both of which can be based on ATP hydrolysis–dependent changes in the geometry of the SMC coiled-coil domains.

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