A joint-ParB interface promotes Smc DNA recruitment

Abstract

Chromosomes readily unlink and segregate to daughter cells during cell division, highlighting a remarkable ability of cells to organize long DNA molecules. SMC complexes promote DNA organization by loop extrusion. In most bacteria, chromosome folding initiates at dedicated start sites marked by the ParB/parS partition complexes. Whether SMC complexes recognize a specific DNA structure in the partition complex or a protein component is unclear. By replacing genes in Bacillus subtilis with orthologous sequences from Streptococcus pneumoniae, we show that the three subunits of the bacterial Smc complex together with the ParB protein form a functional module that can organize and segregate foreign chromosomes. Using chimeric proteins and chemical cross-linking, we find that ParB directly binds the Smc subunit. We map an interface to the Smc joint and the ParB CTP-binding domain. Structure prediction indicates how the ParB clamp presents DNA to the Smc complex, presumably to initiate DNA loop extrusion.

Keywords: CP: molecular biology; DNA loop extrusion; ParABS; ParB; ScpA; ScpB; Spo0J; chromosome segregation; condensin; parS; smc.

Graphical abstract

Figure 1

A four-component system for chromosome organization in bacteria (A) Schematic of Smc recruitment via ParB at parS sites (left) and chromosome organization by DNA loop extrusion (right). Chromosome, chr.; DNA replication forks, forks. (B) Viability assessment of gene-transplanted strains by spotting on nutrient-poor medium (SMG) and nutrient-rich medium (ONA). Gene identity of strains indicated by colored bars, Bsu in blue colors, Spn in orange colors. Spotting was performed in technical triplicates. (C) Normalized 3C-seq contact maps of strains with the indicated genotypes from exponentially growing cultures. Additional maps are shown in Figure S1. All 3C-seq contact maps presented are divided into 10-kb bins with the replication origin being placed in the middle. The interaction score is on a log10 scale (for more details, see STAR Methods). Note that the contact map for the wild type is the same as in Anchimiuk et al. (2021). The 3C-seq experiments were performed in biological duplicates yielding comparable results. See also Figure S1.

Figure 2

Smc binds the ParB N domain (A) Alignment of Bsu and Spn ParB protein sequences. Identical residues are denoted by blue background colors, divergent residues in gray colors. Construction of ParB chimeras is indicated in red colors at the N to M domain transition and in green colors at the M to C domain transition. The black brackets denote ParA-interacting residues that were removed from chimeric constructs harboring N-terminal sequences of Bsu origin. (B) ParB domain structure. Constructions of chimeras are indicated by brackets. (C) Microscopy image of B. subtilis cells harboring ^SpnSmc-ScpAB with the ^Spn198-C ParB chimera fused to GFP protein. See Figure S2C for other chimeras. (D) Spotting assay of B. subtilis strains carrying ^SpnSmc-ScpAB as well as the indicated chimeric ParB proteins, as in Figure 1B. Spotting was performed in technical triplicates. See also Figure S2.

Figure 3

Fine mapping of the Smc-binding site on ParB (A) Sequence alignment of Bsu and Spn ParB N domain, as in Figure 2A. Grouping of ParB residues in patches (1–4, top labels) as well as subgrouping (A, B, and C, bottom labels). (B) Viability assay by dilution spotting for ^SpnSmc-ScpAB strains carrying chimeric ParB proteins, as defined in (A). As in Figure 1B. Spotting was performed in technical triplicates. (C) As in (B) for additional ParB chimeras. (D) Distribution of the identified Smc-interacting residues on the surface of the CTP-engaged ParB N-domain dimer (PDB: 6SDK [Soh et al., 2019]). ParB chains are shown in blue and gray colors, respectively. Key residues are indicated and highlighted in yellow, orange, and brown colors. Notably, the presence of parA significantly reduced the viability of some of the strains, indicating that ParA mis-regulation is not tolerated well by these ParB variants when also combined with ^SpnSmc-ScpAB (Figure S3A). See also Figure S3.

Figure 4

The Smc joint domain targets ParB (A) Left: Schematic of Smc-ScpAB and the structure of the Bsu Smc joint (PDB: 5NMO) denoting the construction of chimeric Smc proteins, blue colors indicating Bsu sequence identity, orange colors indicating Spn origin. Right: Chromatin immunoprecipitation coupled to quantitative PCR (ChIP-qPCR) using α-ScpB serum undertaken with chimeric Smc strains as denoted. See additional experiments in Figure S4C. (B) Left: Schematic and structural model of Smc protein; displayed as in (A). Right: Viability assay by spotting strains carrying ^Spnjoint in combination with the indicated ParB chimeras. As in Figure 1B. Spotting was performed in technical triplicates. For the corresponding ChIP-qPCR results, see Figure S4D. See also Figure S4.

Figure 5

In vivo cross-linking of ParB and Smc proteins. (A) Schematic of chemical cross-linking by the heterobifunctional molecule SMCC. (B) Candidate ParB cysteine residues and their position (in red colors) on the ParB-CDP dimer (in surface representation with chains in blue and gray colors). (C) SMCC cross-linking using ParB(Cys) mutants as indicated and detected by in-gel fluorescence detection of Smc(mH/EQ)-HT (“Smc-HT”) protein. Higher molecular weight species appearing upon cross-linking are indicated by asterisks. Cross-linking was performed in technical duplicates. (D) Schematic of BMOE cross-linking chemistry (top) and candidate Smc(Cys) residues and their distribution on the Smc joint structure (cartoon representation). (E) BMOE cross-linking using combinations of ParB(Cys) and Smc(Cys) mutants as indicated. Samples were enriched for ParB interacting material by incubation with α-ParB antibody coupled Dynabeads. Detection by in-gel fluorescence of Smc(mH/EQ)-HT (“Smc-HT”) protein. Cross-linked ParB-Smc species are indicated by asterisks. Cross-linking was performed in technical duplicates. (F) Positioning of identified cross-linking residues on ParB (left, as in [B]) and Smc (right, Smc joint in the rod configuration in surface representation). See also Figure S5.

Figure 6

Prediction and evaluation of the joint-ParB interface structure (A) Reconstruction of a Smc-ParB sub-complex by superimposition of the crystal structure of a ParB NM domain dimer (PDB: 6SDK) with a joint-ParB heterodimer predicted by AF-Multimer in surface representation in side view (top) and top view (bottom). The Smc chain is displayed in gray colors, and the ParB chains are displayed in dark and light blue colors, respectively. (B) The Smc-ParB sub-complex shown in cartoon representation with residues used for cysteine cross-linking experiments highlighted as sticks in red colors. C_α-C_α distances (in Å) are indicated by dashed lines. (C) Same as in (B) with Smc and ParB residues identified by genetic sequence matching displayed in red colors. (D) Mutagenesis of selected residues (marked in stick representation) at the Smc-ParB interface. The predicted structure of the Smc joint (in gray colors) and the ParB NM domains (in marine colors) are displayed. The nucleotide CDP is shown in stick representation. Mutations that were pursued further are denoted in orange colors, other residues in olive colors. (E) Viability assay by dilution spotting for candidate strains harboring Smc-binding interface mutants of ParB in smc-pk3 and Δsmc backgrounds. Spotting was performed in technical triplicates. (F) Immunoblotting to compare cellular levels of wild-type (WT) and mutant ParB using antiserum raised against Bsu ParB. (G) Deep-sequencing DNA profiles of parB(L69S,K70E,E101K) and ΔparB input samples normalized to the DNA profile of WT cells. For bins with read counts greater than the WT sample, the ratio was plotted above the genome coordinate axis (in blue). Otherwise, the inverse ratio was plotted below the axis (in orange). Additional representations of the data are included in Figure S6. (H) As in (G) but displaying ChIP efficiency (reads in IP/reads in Input) normalized to the ChIP efficiency obtained with WT cells. Ratios greater than 1 are displayed in blue, for other bins, the inverse ratio is shown in orange. ChIP-seq was performed on two biological replicates with comparable outcomes. Additional representations of the data are available in Figure S6. See also Figure S6.

Figure 7

Structural similarities and models (A) Comparison of the joint-ParB interface (left panels) and the Smc3 joint-Scc2 interface in human cohesin (right panels). ParB-proximal and Scc2-proximal residues on the Smc and Smc3 joint (C_α-C_α distance <10 Å), respectively, are indicated in red colors. The Scc2/DNA structure in ATP-engaged human cohesin is displayed (PDB: 6YUF) (right panel). Only the Smc3 and Scc2 subunits are shown for simplicity. For direct comparison, the SMC subunits are also displayed in isolation. Superimposition with MukB (PDB: 7NZ3) was omitted due to significant structural divergence of the respective joint domains (Bürmann et al., 2021). (B) Putative models for the contact between ParB-clamped DNA and the Smc dimer. ScpAB is omitted from some representations for simplicity. Two scenarios are considered: DNA passage between disengaged heads (“1”) and insertion of a DNA loop into the Smc interarm space (“2”), the product of which are shown on the left and right panels, respectively. ParB is shown to interact with the right Smc monomer (ParB in “front” of Smc). ScpAB can either associate on the same side (“front”) or on the other side (“back”). Possible variations of these scenarios with pseudo-topological and non-topological modes of DNA association are not shown for the sake of simplicity. See also Figure S7.