Control of Smc Coiled Coil Architecture by the ATPase Heads Facilitates Targeting to Chromosomal ParB/parS and Release onto Flanking DNA

Abstract

Smc/ScpAB promotes chromosome segregation in prokaryotes, presumably by compacting and resolving nascent sister chromosomes. The underlying mechanisms, however, are poorly understood. Here, we investigate the role of the Smc ATPase activity in the recruitment of Smc/ScpAB to the Bacillus subtilis chromosome. We demonstrate that targeting of Smc/ScpAB to ParB/parS loading sites is strictly dependent on engagement of Smc head domains and relies on an open organization of the Smc coiled coils. We find that dimerization of the Smc hinge domain stabilizes closed Smc rods and hinders head engagement as well as chromosomal targeting. Conversely, the ScpAB sub-complex promotes head engagement and Smc rod opening and thereby facilitates recruitment of Smc to parS sites. Upon ATP hydrolysis, Smc/ScpAB is released from loading sites and relocates within the chromosome-presumably through translocation along DNA double helices. Our findings define an intermediate state in the process of chromosome organization by Smc.

Graphical abstract

Figure 1

Smc ATPase Activity Determines the Chromosomal Distribution of Smc/ScpAB (A) Schematic representation of the Smc ATPase cycle. (B) Immunoblotting of ATPase mutant Smc proteins with α-Smc antiserum. Whole-cell extracts from strains BSG1002, BSG1007–BSG1008, BSG1067, BSG1045–BSG1047, and BSG1083. See also Figure S1B. (C) Fluorescence images of cells harboring mGFP-tagged Smc alleles: BSG1002, BSG1067–BSG1068, BSG1855–BSG1857, and BSG1881. Scale bar, 2 μm. Differential interference contrast (DIC) (bottom) and GFP fluorescence images (top) are shown. Quantification of foci number per cell is given in Figure S1H. (D) ChIP-qPCR analysis of cells from strains BSG1002, BSG1007–BSG1008, BSG1045–BSG1047, and BSG1083 using α-Smc antiserum. Error bars were calculated from two independent experiments as SD. Please note that values of ChIP enrichment below and above 0.06% are displayed on different scales given on the left and right side of the graph, respectively. The analysis of chromosomal loci harboring highly transcribed genes (such as the tRNA cluster trnS) generally produce ambiguous results with relatively high levels of ChIP signal in control samples (Δsmc). This seems to be a widely observed phenomenon in ChIP and it remains unclear whether the enrichment is physiologically relevant. See also Figure S1.

Figure 2

Hydrolysis Mutant but Not Wild-Type Smc Co-localizes with ParB/parS (A) Close-up view of ChIP-seq profiles for wild-type Smc (BSG1002) (top panel) and Smc(EQ) (BSG1008) (bottom panel) generated using antiserum raised against the Bs Smc protein. Sequence reads were mapped to 1 kb windows spaced at 100-bp intervals and normalized for input DNA as follows. The number of reads for the ChIP sample in a given window was divided by the number of reads in the input sample for the same window (after normalizing the total number of reads). Raw input and ChIP data are shown in Figure S2. Axes labeled in green color highlights different scaling. Asterisks indicate the positions of parS sites. (B) Close-up view of the ChIP-seq profile of ParB protein (from BSG1470 cells) generated using antiserum raised against purified BsParB-His6 protein. Data analysis and presentation as in (A). (C) Whole-genome views of data presented in (A). Sequence reads were mapped to 5-kb windows spaced at 5-kb intervals across the genome and normalized for input DNA. (D) To highlight differences between the distribution of wild-type Smc and Smc(EQ) the normalized ratios for Smc(EQ) in a given window was divided by the equivalent ratio for Smc(wt). Numbers above one are shown in yellow colors (axis on the left side). For numbers below one, the inverse ratio was calculated and displayed in gray colors (axis on the right side). (E) Whole-genome view of the ParB ChIP-seq data presented in (B). Data analysis as in (C). See also Figure S2.

Figure 3

Dimerization at the Smc Hinge Hinders Chromosomal Association of Smc/ScpAB (A) ChIP-qPCR was performed on exponentially growing cells of strains BSG1051–BSG1052, BSG1406, and BSG1387 using α-Smc antiserum. Quantification of input and eluate material was done by qPCR using primer pairs specific for the indicated loci. (B) As in (A) using strains BSG1889–BSG1894. (C) The scheme indicates the disruptive effect of mutations in the Smc hinge domain on dimerization. ChIP-qPCR was performed with α-Smc antiserum on strains BSG1620–BSG1621, BSG1624, BSG1890, and BSG1892–BSG1893. (D) Fluorescence imaging of Smc-mGFP fusion proteins in cells of strains BSG1067–BSG1068, BSG1378, BSG1413, BSG1662, BSG1677, and BSG1798–BSG1799. Scale bar, 2 μm. Quantification of foci number per cell is given in Figure S4C. Same experiments as in Figure 1C. See also Figure S3.

Figure 4

Hinge Dimerization and Head Engagement Control the Conformation of Smc/ScpAB (A) Structure of ATP engaged Pf Smc head domains (PDB: 1XEX) in brown and pink colors, respectively, (bottom view). Residue D1131 is indicated in ball representation in orange colors (middle panel). The distance between the carboxyl carbon atom in the side chains of the D1131 symmetry mates is estimated to be ∼6 Å (right panel). A sequence alignment between PfSmc and BsSmc shows that K1151 in BsSmc corresponds to D1131 in PfSmc. (B) In vivo BMOE crosslinking of Smc(K1151C)-HaloTag in cells of strains BSG1488, BSG1509, BSG1512, BSG1547, BSG1597–BSG1598, BSG1791, and BSG1800. Four endogenous cysteine residues were replaced by serines. Cross-linked Smc-Halotag species were detected by in-gel fluorescence of the HaloTag-TMR substrate (left panel). Smc^∗ indicates a degradation product of Smc(mH). The graph (right panel) shows mean values and SDs from three replicates. (C) Same as in (B) using A715C as sensor cysteine for formation of Smc rods by the hinge proximal Smc coiled coil. In vivo crosslinking of Smc(A715C) with bismaleimidoethane (BMOE) in strains BSG1921–BSG1924, BSG1949–BSG1951, and BSG2036. T test statistics: ^∗∗∗p ≤ 0.001; not significant (n.s.), p > 0.05. See also Figure S4.

Figure 5

A Large Central Part of Smc Is Dispensable for Targeting to parS (A) ChIP-qPCR was performed with TAP-tagged alleles of Smc using IgG-coupled magnetic beads for immunoprecipitation. Strains: BSG1671–BSG1672, BSG1689, BSG1691, BSG1779–BSG1780, and BSG1895–BSG1896. The schemes on top represent modifications to the Smc hinge in Smc(mH) (left) and Smc(ΔH) (right). (B) Schematic overview of the series of internal Smc truncation constructs. Solid and dashed horizontal lines denote the presence and absence of Smc sequences in a given truncation construct. A gray box demarcates the central portion of the Smc protein, which is dispensable for targeting to parS-359. N-terminal and C-terminal Smc sequences are fused via a short peptide linker (-GGGSGGGSGGG-). The name of a given truncation construct indicates the predicted length of its Smc coiled coil. Labels in green and red colors indicate efficient and inefficient targeting to parS. All proteins are tagged with a TAP tag at their C terminus. Purple vertical lines and boxes indicate disruptions in the Smc coiled coil (Waldman et al., 2015). (C) ChIP-qPCR against the TAP tag of strains BSG1520, BSG1689, BSG1779, BSG1825, BSG1871–BSG1872, and BSG1874–BSG1875 (left panel). Immunoblot against the TAP tag with strains BSG1002, BSG1016, BSG1475, BSG1520, BSG1689, BSG1779, BSG1825, BSG1871–BSG1872, and BSG1874–BSG1875 (right panel). (D) Same as in (C) with another set of Smc truncation constructs. ChIP-qPCR with strains BSG1779, BSG1824, BSG1826–BSG1830 and BSG1873 (left panel). Anti-TAP immunoblot with strains BSG1002, BSG1016, BSG1475, BSG1779, BSG1824, BSG1826–BSG1830, and BSG1873 (right panel). See also Figure S5.

Figure 6

Smc/ScpAB Relocates from parS Loading Sites to Distant Parts of the Chromosome upon ATP Hydrolysis (A) ChIP-seq using α-ScpB antiserum on strains BSG1002 (parB) (top panel) and BSG1052 (ΔparB) (middle panel). Reads were mapped to 5-kb bins. Signals for IP samples were divided by the signals of the normalized input. Ratios were calculated by dividing the values obtained for the wild-type strain by the numbers of the ΔparB strain (bottom panel). All values above one are shown in orange colors. For all other windows the inverse ratio was calculated and displayed in gray colors. (B) ChIP-seq using α-ScpB antiserum on strains BSG1470 (mparS-amyE) (top panel) and BSG1469 (parS-amyE) (middle panel). Reads were mapped to 5-kb bins. Signals for IP samples were divided by the signals of the normalized input. Ratios were calculated by dividing the values of the parS-amyE strain by the mparS-amyE strain (bottom panel). A number above one indicates more reads in the parS-amyE sample (shown in the blue colors), for all other windows, the inverse ratios were calculated and displayed in gray colors. See also Figure S6.

Figure 7

Model for the Recruitment of Smc/ScpAB to and Release from parS Sites (A) Model for the targeting to and release from ParB/parS by holo-Smc/ScpAB. Most Smc/ScpAB exists as a rod-shaped structure, which is unable to bind to DNA via its hinge or to ParB/parS via the coiled coils. Dissolution of the Smc rod and engagement of Smc head domains are prerequisites for the targeting of Smc/ScpAB to parS. Upon ATP hydrolysis, the ring-like structure might revert to the rod conformation and is released from parS DNA. Sister DNA segments (in green colors) might be excluded from the Smc rod due to steric restrictions. Repetitive rod-ring-rod transitions might drive DNA loop extrusion. (B) Pie charts displaying rough estimates for the relative occupancy of the different states illustrated in (A) based on Smc head cross-linking efficiency (Figure 4B). Please note that the fraction of wild-type Smc complexes on and off the chromosome (depicted as green and gray pies in the left chart) is unknown. A tiny fraction of chromosomally loaded Smc(EQ)/ScpAB has been detected (Wilhelm et al., 2015).