Clamping of DNA shuts the condensin neck gate

Abstract

SignificanceDNA needs to be compacted to fit into nuclei and during cell division, when dense chromatids are formed for their mechanical segregation, a process that depends on the protein complex condensin. It forms and enlarges loops in DNA through loop extrusion. Our work resolves the atomic structure of a DNA-bound state of condensin in which ATP has not been hydrolyzed. The DNA is clamped within a compartment that has been reported previously in other structural maintenance of chromosomes (SMC) complexes, including Rad50, cohesin, and MukBEF. With the caveat of important differences, it means that all SMC complexes cycle through at least some similar states and undergo similar conformational changes in their head modules, while hydrolyzing ATP and translocating DNA.

Keywords: DNA motor; SMC complex; cryo-EM; loop extrusion.

Fig. 1.

(A) Architecture of the yeast condensin SMC complex. Adding DNA and an ATP analog leads to the formation of the clamped state, which is the subject of this study. The dotted rectangle marks the portion of the head module in the clamped state, of which the cryo-EM structures were solved. (B) Two-dimensional class averages of cryo-EM images of the condensin tetramer, containing subunits Smc2, Smc4, kleisin Brn1, and Ycs4. The dsDNA is clearly visible, whereas the coiled-coil arms of Smc2 and Smc4 and the hinge domains are highly flexible with respect to the well-resolved head module. (C) A 2.95-Å resolution cryo-EM map of the clamped condensin head module bound to DNA (Form I). (D) Cartoon representation of the atomic model built into and refined against the map shown in C. Various parts of the kleisin Brn1 are disordered.

Fig. 2.

(A) Top view of the pseudo-two-fold symmetric ATPase head domains of Smc2 and Smc4. Two molecules of ADP.BeF₃ are sandwiched between them, leading to the engagement of the SMC head domains. (B) The clamped DNA binds to the largely positively charged upper surface of the ATPase head domains of Smc2/4 but is not perfectly aligned with the pseudo-two-fold axis. The DNA also makes contacts with the coiled-coil neck of Smc2 and Brn1, but not the neck of Smc4. (C) In the clamped head module, Ycs4 makes extensive contacts with the DNA via a positively charged groove toward the N terminus and through a small contact near the C terminus. (D) Ycs4 make four contacts with other subunits within the head module: (E) with the Smc2 neck and Brn1, (F) with a loop emanating from the Smc2 head domain that also contacts Brn1 as it binds to Ycs4, (G) with a large patch on the back surface of Smc4, and (H) with an unstructured and poorly conserved part near the N terminus of Smc4.

Fig. 3.

(A) Architecture of the condensin pentamer in the clamped state, containing subunits Smc2, Smc4, Brn1, Ycs4, and Ycg1. (B) Cryo-EM map at 3.2-Å resolution of Ycg1 alone, bound to DNA, obtained from a dataset collected on the condensin pentamer, presumably because Ycg1 is not rigidly attached to the complex or head module. (C) Cartoon representation of the atomic model built into and refined against the map shown in B. The “safety belt” (21) formed by Brn1 is well-resolved, except for one disordered loop. (D) Proposed path of the kleisin Brn1 through the clamped state of yeast condensin. Several regions could not be resolved in our cryo-EM map (Fig. 1C), but the location of ordered sections suggests the kleisin remains above the clamped DNA, leading to E-K entrapment. Large Brn1 loops connect Ycg1 to the head module, which enables Ycg1 to reside at a distance as shown in Fig. 4A.

Fig. 4.

(A, Left and Middle) Condensin pentamer clamping circular plasmid DNA, as observed by cryo-EM with a VPP. Ycg1 bound to DNA is at a distance from the head module, presumably because Ycg1 is flexibly attached via Brn1. (A, Right) Schematic drawing and superimposed 2D class averages of the image in the middle, highlighting that a DNA loop between the head module and Ycg1 has formed (dark gray). The opened SMC coiled-coil arms are just about visible. (B) The condensin head module clamping circular DNA adopts the same conformation as on linear DNA (compare with Fig. 1C). (C) Equally, Ycg1 binds to circular DNA in a manner similar to linear DNA (compare with Fig. 3B). (D) The clamped structure of the head module is compatible with two different paths of the DNA with respect to the SMC arms and hinge domains, which are not resolved, leading to different loading paths for topological entrapment. E-K + E-S requires the DNA to pass through the nonengaged heads, whereas E-K + S-K requires opening of a gate in the tripartite S-K ring Smc2–Smc4–Brn1 (SI Appendix, Fig. S1A). (E, Left) The Smc2–Brn1 neck gate is closed in the clamped state of condensin. The neck gate involves the N-terminal helical domain of Brn1 binding to the coiled-coil neck of Smc2. An N-terminal portion of Ycs4 and also the clamped DNA may be there to stabilize the neck gate that closes the tripartite S-K ring consisting of Smc2, Smc4, and Brn1. (E, Right) Brn1 N-terminal release assay. Engineered TEV cleavage sites enable the N-terminal Brn1 domain to be cleaved and washed away from beads as long as the neck gate is not closed. The N-terminal fragment was labeled with a fluorescent dye (LD555) for visualization. In the absence of DNA, addition of ATP or ADP.BeF₃ opens the gate, while the simultaneous presence of DNA keeps the gate shut, presumably through the formation of the clamped state of condensin as shown here.