Tuned SMC Arms Drive Chromosomal Loading of Prokaryotic Condensin

Abstract

SMC proteins support vital cellular processes in all domains of life by organizing chromosomal DNA. They are composed of ATPase "head" and "hinge" dimerization domains and a connecting coiled-coil "arm." Binding to a kleisin subunit creates a closed tripartite ring, whose ∼47-nm-long SMC arms act as barrier for DNA entrapment. Here, we uncover another, more active function of the bacterial Smc arm. Using high-throughput genetic engineering, we resized the arm in the range of 6-60 nm and found that it was functional only in specific length regimes following a periodic pattern. Natural SMC sequences reflect these length constraints. Mutants with improper arm length or peptide insertions in the arm efficiently target chromosomal loading sites and hydrolyze ATP but fail to use ATP hydrolysis for relocation onto flanking DNA. We propose that SMC arms implement force transmission upon nucleotide hydrolysis to mediate DNA capture or loop extrusion.

Keywords: ParB; SMC; Smc/ScpAB; chromosome segregation; cohesin; coiled coil; condensin; heptad repeat; kleisin; periodicity.

Graphical abstract

Figure 1

Coiled-Coil Length Distributions of SMC Proteins (A) Subunit composition of SMC-kleisin complexes. (B) Coiled-coil length distribution of prokaryotic (Pr; n = 3,337) and eukaryotic (Eu; n = 1,659) SMC sequences. Arm lengths were estimated on the basis of multiple sequence alignments. The dashed line indicates the coiled-coil length of Bs Smc. (C) Kernel density estimates for data shown in (B). Dashed lines indicate positions of prominent modes. (D) Arm length distribution for eukaryotic SMC sequences classified by type of complex. See also Figure S1.

Figure 2

High-Throughput Screens for Functionally Resized Smc (A) Strategy for an arm truncation screen. Smc arms were shortened by grafting the hinge and a short stretch of hinge-proximal coiled coil onto a shortened head-proximal part (left). An arrow illustrates the tested size-range. Shortened alleles were assembled by a Golden Gate approach (right). (B) Strategy for an arm extension screen. Smc arms were either extended or shortened by resizing the Bs part of a functional chimeric Bs/S. pneumoniae (BsSp) protein. As in (A). (C) Viability of strains with resized Smc arms. Modified smc alleles were transformed into smc-null cells for allelic replacement at the endogenous locus. Transformation mixtures were plated on ONA with antibiotics, and growth was assessed by the total area of bacterial colonies per plate. Truncation and extension screen were performed independently and normalized to their respective 95% growth quantile. (D) Power spectrum of data shown in (C). The periods of the major peaks are indicated. (E) Dilution spotting of strains with short smc alleles. Strains were constructed on SMG in the absence of selection pressure for smc function. Strains were spotted either on rich (ONA) or minimal (SMG) medium. Expression of the engineered alleles was probed by western blotting using polyclonal antibodies raised against full-length Smc. Note that modification of the Smc protein possibly removes some of the epitopes. Coomassie staining of extracts run on a separate SDS-PAGE gel is shown as a loading control. CBB, Coomassie Brilliant Blue; CC, coiled coil. (F) Dilution spotting of strains with long smc alleles. As in (E). See also Figure S2.

Figure 3

Dimerization and ATPase Activity of Mini-Smc Proteins (A) In vivo site-specific crosslinking of Mini-Smc variants at the hinge interface (see also Figure S2B). In-gel fluorescence after BMOE crosslinking of strains containing cysless Smc-HaloTag variants (top) and quantification thereof (bottom) is shown. Crosslinking was performed in three separate reactions. Colored boxes indicate 95% credible intervals, horizontal lines indicate mean and SD of the data. (B) Head engagement levels in Mini-Smc proteins monitored by in vivo site-specific crosslinking at the reporter residue K1151C (see also Figure S3C) (Lammens et al., 2004, Minnen et al., 2016). The SR mutation blocks head engagement, and the EQ mutation blocks ATP hydrolysis (Figure S3A). As in (A). (C) Purification of Smc variants. Purified fractions were analyzed by SDS-PAGE and Coomassie staining. KI, Smc ATP-binding mutation K37I. (D) Steady-state ATPase activity of purified Smc variants at 0.3 μM protein and variable ATP concentration. Activity was determined by a coupled enzyme assay and data were fitted by the Hill model (see also Table S3). Data and fits for three replicates are shown. See also Figure S3.

Figure 4

Chromosomal Loading of Smc-ScpAB Containing Mini-Smc Proteins (A) ChIP-seq profiles at the oriC region. ChIP was performed with an antiserum raised against ScpB. Normalized counts in reads per million (rpm) are plotted against the distance from oriC (top). The bottom graph shows the ratiometric analysis against the wild-type profile. For each bin, normalized counts were compared with the respective wild-type value. The higher value was divided by the lower. For bins where the mutant value was greater than or equal to the wild-type value, the ratio was plotted above the genome coordinate axis (blue) and below the axis otherwise (orange). EQ, Smc(E1118Q). (B) ChIP-qPCR against ScpB for mini-smc strains containing an ATPase mutation that prevents head engagement (SR, S1090R). Loci close to parS sites are colored in blue, loci close to the replication origin are orange, and chromosomal arm positions are white (see Figure 4A, top). (C) Chromosome entrapment assay for strains containing Mini-Smc complexes. Smc-ScpAB complexes containing Smc-HaloTag variants were site-specifically cross-linked at hinge and ScpA-Smc interfaces and conjugated to a HaloTag-OregonGreen (OG) substrate. Intact chromosomes were isolated in agarose plugs, and proteins were extracted under denaturing conditions. Smc-HaloTag species retained in the plug were resolved by SDS-PAGE and detected by in-gel fluorescence. Species a–g and i are linear, species h/h′ are circular. See also Figure S4.

Figure 5

Suppressor Mutagenesis and Hinge Replacement of Mini-Smc Variants (A) Suppressor mutations mapped onto the crystal structure of the T. maritima (Tm) hinge domain (Protein Data Bank [PDB]: 1GXL). Bs residues are indicated in black, Tm homologs are indicated in red. (B) Mutations in the arm suppress lethality of the CC320 mini-smc allele. The cartoon illustrates the position of the suppressor mutations (left). The panel on the right shows spot dilutions as in Figure 2D. (C) Comparison of open conformations of the SMC hinge (left; PDB: 1GXL) and the Rad50 Zinc hook (right; PDB: 1L8D). The N-terminal coiled-coil strands are colored green, and the C-terminal strands are colored orange. The part that was substituted to construct a functional Smc(Zh) chimera is shown in blue and gray, respectively. (D) Coiled-coil truncation screen of the Smc(Zh) protein. Data are compared with the arm shortening experiment shown in Figure 2C. The growth axis for Smc(Zh) has been inverted for clarity. As in Figure 2C. (E) Spot dilutions and western blot analysis of Smc(Zh) variants with short coiled coils. As in Figure 2E. See also Figure S5.

Figure 6

Transposon Screen for Functional Smc Variants Containing a Randomly Inserted Peptide (A) Peptide insertion screen. The cartoon on top illustrates the region that was targeted by transposon mutagenesis of a smc-targeting construct. The obtained insertion library was characterized by deep sequencing, and reads containing the insert were selected. Insertion read counts for positions with at least one detected insertion are shown (top). After transformation of the library into a smc-null strain, viable clones isolated on ONA were characterized by Sanger sequencing. Counts of insert positions among viable isolates are shown (bottom). Green regions delineate the head domain, orange delineates the hinge region, and the blue graph indicates coiled-coil probability. (B) Spot dilutions of strains with designed peptide insertions in the coiled-coil arm. As in Figure 2E. (C) ChIP-qPCR against ScpB for strains containing peptide insertions in the Smc arm. Loci close to Smc loading sites are colored in blue, loci close to the replication origin are orange, and chromosomal arm positions are white (see Figure 4A). (D) ATPase activity of non-functional Smc variants with peptide insertions in the coiled-coil arm. As in Figure 3D. See also Figure S6.

Figure 7

Models for the Role of the Coiled-Coil Arm during DNA Transactions of SMC (A) Tentative model for the effect of arm length variation on Smc function. Proteins with a large offset in the super-helical phase of their coiled coils (“Out-of-Tune” complexes) react differently to mechanical strain induced during their ATPase cycle. (B) Models for Smc arm function during chromosomal DNA transactions. After initial recruitment to the chromosome induced by ATP binding, the coiled-coil arms of Smc transduce mechanical energy to open a DNA entry gate (top middle) or directly act on DNA, for example during loop extrusion (bottom middle).