The Smc5/6 Core Complex Is a Structure-Specific DNA Binding and Compacting Machine

Abstract

The structural organization of chromosomes is a crucial feature that defines the functional state of genes and genomes. The extent of structural changes experienced by genomes of eukaryotic cells can be dramatic and spans several orders of magnitude. At the core of these changes lies a unique group of ATPases-the SMC proteins-that act as major effectors of chromosome behavior in cells. The Smc5/6 proteins play essential roles in the maintenance of genome stability, yet their mode of action is not fully understood. Here we show that the human Smc5/6 complex recognizes unusual DNA configurations and uses the energy of ATP hydrolysis to promote their compaction. Structural analyses reveal subunit interfaces responsible for the functionality of the Smc5/6 complex and how mutations in these regions may lead to chromosome breakage syndromes in humans. Collectively, our results suggest that the Smc5/6 complex promotes genome stability as a DNA micro-compaction machine.

Keywords: DNA compaction; DNA repair; SMC; Smc5/6 complex; chromosome; genome stability; supercoiled DNA.

Figure 1.. Purification of the Smc5/6 complex.

(A) Schematic representation of the Smc5/6 complex. (B) Overview of the 2 yeast strains used for Smc5/6 complex overexpression and purification. Each strain contains 2 plasmids; one for the overexpression of the Nse4/SMC subunits and the other –common to both strains– that overexpresses Nse1 and Nse3. (C) Representative gel showing the different chromatography steps used to purify the Smc5/6 dimer. The positions of StreptagII-Smc6 and His-Smc5 are shown on the right. (D) Purification steps used to isolate the Smc5/6 core complex. The positions of individual proteins prior to TEV cleavage are shown on the left of the gel, while the positions of subunits after TEV-induced removal of purification tags/linker are shown on the right of the gel. After completion of the TEV cleavage reaction, all the subunits of the Smc5/6 complex migrate in SDS-PAGE at the positions of the native full-length proteins. Note that tag-less Nse4a migrates at the same apparent mass as GST-Nse1 (i.e., prior to TEV-induced removal of the GST tag). Likewise, TEV and tag-less Nse1 are co-migrating on the gel, but TEV is removed from the Smc5/6 complex at the final gel exclusion step. (E) Analysis of the oligomerization status of the Smc5/6 core complex by density gradient centrifugation. See also Figures S1–3.

Figure 2.. Functionality of Smc5/6 complexes containing Nse4-SMC fusions.

(A) Phenotype of SMC5/6-NSE4 fusion alleles after sporulation. One copy of NSE4 and either SMC5 or SMC6 were deleted in diploid yeast strains expressing Nse4-linker-Smc5 or Smc6-linker-Nse4 fusion proteins. The linker sequence and fusion strategy employed in these strains are identical to those used to purify the human subunits, except that yeast Smc5, Smc6 and Nse4 protein sequences were used to allow complementation of nse4∆, smc5∆ and smc6∆ deletions. Diploid strains of the indicated genotype were sporulated and haploid spores micromanipulated on solid growth medium. The viability of spores was scored after 3 days of germination, and their genotypes are represented schematically under the growth plates. –/– and NSE4-L-SMC/– indicate the absence or presence of fusion alleles at the URA3 locus of parental/diploid yeast. (B-C) Proliferation capacity of two independent smc5∆ nse4∆ clones (MATa and MATα) expressing the Nse4-linker-Smc5 fusion protein was assessed by serial dilution assay on solid medium. After plating, cells were grown for 2–3 days at the indicated temperatures (23 °C, 30 °C and 37 °C; panel B) in the absence or presence of DNA damaging drugs (HU, MMS or 4NQO; panel C).

Figure 3.. Shape and configuration of the Smc5/6 dimer and core complex.

(A) Stabilization of the Smc5/6 core complex using the GraFix procedure (Kastner et al., 2008). In the absence of crosslinking, subunits of the complex eluted as individual bands in sucrose density gradients, whereas they eluted as a single band of high molecular mass after GraFix crosslinking. (B-C) Representative single particles of Smc5/6 core complexes prior (B) and after (C) GraFix treatment. Box dimensions are 518 Å x 518 Å. (D) Single particle images showing the Smc5–6 heterodimer after GraFix stabilization. (E-F) Two-dimensional class averages of Smc5/6 core particles pre- (E) and post-GraFix treatment (F). Dimensions are represented in Å and images in E and F were reconstituted from 44 and 68 particles, respectively. (G) Two-dimensional class average of GraFix-stabilized Smc5/6 dimer (n = 167 particles). (H) Proximity maps showing intra- and inter-subunit connections within the Smc5/6 core complex. XLs identified in the MS analysis were plotted on circular diagrams corresponding to the amino-acid sequences/functional domains of Smc5/6 complex subunits. Intra-molecular connections are shown as grey lines, whereas inter-molecular contact points are shown in the inner part of the diagram (blue lines are for XLs specific to CC domains; orange lines are for XLs connecting hinge domains; green lines are for all other XLs). (I) Network of inter- and intra-subunit connection points within the Smc5/6 dimer. See also Figures S3–S4 and Table S1.

Figure 4.. Impact of mutations in the WH domain of Nse3 and the ATPase/CC neck of Smc6.

(A) Schematic representation of Nse3 domain structure. The positions of LIC syndrome mutations are marked with asterisks (van der Crabben et al., 2016). (B) Alignment of the WH-B extension domain in eukaryotic homologs of Nse3. (C) Crystal structure of the Nse3-Nse1 dimer. The position of mutations created in this study is marked with yellow ovals in the magnified view (inset). The structure is PDB 3NW0 from Newman et al. (2016). (D) Proliferation phenotype of yeast strains carrying mutations in Smc5/6 complex components. Strains were diluted on solid medium containing DNA damaging agents and grown at the indicated temperatures for 2–4 days before recording their phenotype. (E) Effect of temperature on the stability of Smc6 mutants. Cell lysates of yeast grown at the indicated temperatures were resolved by SDS-PAGE and processed for immunoblot analysis. The positions of Smc6 and loading control (*) bands are shown on the right. (F) Quantitative analysis of Smc6 abundance determined by immunoblot. The bar graph reports the mean protein abundance of Smc6-R135E/R144E relative to wild-type Smc6 ± SEM for 3 independent experiments. * signifies p-value ≤ 0.05 and ** a p-value ≤ 0.01 (ANOVA with Dunnett’s post hoc test).

Figure 5.. DNA-binding affinity and substrate preference of the Smc5/6 core complex.

The DNA-binding behavior of the Smc5–6 dimer (A) and core complex (B-C) was determined by EMSA saturation experiments using various DNA substrates. Purified complexes were incubated with the indicated nucleic acids for 30 min at 30 °C and the resulting protein-DNA complexes were resolved by electrophoresis. The molar excess of Smc5/6 complex over DNA is shown above each lane, whereas the positions of unbound and Smc5/6-bound DNA substrates are marked by arrowheads and asterisks, respectively. The graphs next to the agarose gels show the quantification of DNA-binding activity. The data reported in the graphs is the mean DNA binding ± SE of ≥3 independent experiments. See also Figure S5A.

Figure 6.. Compaction of DNA by the Smc5/6 complex.

(A) Schematic representation of magnetic tweezers single-DNA compaction assay. (B) DNA extension measured following addition of 5 nM Smc5/6 complex (added slightly before t=0) with no nucleotide. As force was reduced from 2 pN to 0.3 pN, there was no compaction observed; the reduction of length is just that expected from thermal bending fluctuations of DNA, also responsible for the increased Brownian motion around the stable average DNA extension. (C) DNA extension following addition of 5 nM Smc5/6 complex (at t = 0) plus 1 mM ATP (separate curves are from separate experiments). At each force, compaction occurred, with more rapid and more complete compaction at lower forces. (D) Compaction rate in the presence of 1 mM ATP at 0.5 pN force measured from a series of 4 experiments of the type shown in panel C for each force (note logarithmic rate scale). A rapid increase in compaction rate with decreasing force (essentially shut off above 1 pN) was observed, indicative of a DNA-loop-capture process of compaction. (E) Total compaction (ratio of change in extension to initial extension) in a series of 4 experiments at different forces, indicating that compaction is more complete at lower forces, and essentially shut off above 1 pN. (F) Total compaction at 0.5 pN in the presence of various nucleotides (0 and 1 mM ATP, 1 mM ADP, and ATPγS). All error bars indicate SEM.

Figure 7.. Capture of DNA by the Smc5/6 core complex requires supercoiling and ATP.

(A) Schematic representation of the reaction steps in the plectoneme/supercoil capture experiment conducted under increased salt conditions. (B) At 100 mM potassium glutamate and 1 mM ATP, a 10 kb DNA under 0.3 pN force has stable extension (purple points, left); following increase of linking number to ∆Lk = −30 (blue), the molecule forms plectonemic supercoils (dark green points); when linking number is returned to ∆Lk = 0, only part of the DNA length contained within the plectoneme is returned (purple points, right); about 1000 nm of length has been “captured” by the Smc5/6 core complex. (C) Experiment similar to B showing capture of supercoiled DNA by the Smc5/6 core complex following supercoiling to ∆Lk = +30 and return to ∆Lk = 0. (D) In the absence of ATP, linking number can be reversibly and repeatedly cycled between ∆Lk = 0, +30 and −30 with no capture of supercoiling by the Smc5/6 complex. (E) Averages of capture experiments over a series of 5 trials show that a large fraction of the DNA length change resulting from plectonemic supercoiling can be captured by the Smc5/6 core complex in the presence of ATP (i.e., right bars; equivalent to step 4 in panel A). Changes observed in DNA extension/length are expressed as plectoneme size (nm) in the graph. The initial size of plectonemes formed with naked DNA (left bars; equivalent to step 2 in panel A) or in the presence of 5 nM Smc5/6 core complex (central bars; step 3) are shown for comparison. Each bar shows mean and SEM. See also Figure S5B,C.