In Vivo Evidence for ATPase-Dependent DNA Translocation by the Bacillus subtilis SMC Condensin Complex

Abstract

Structural maintenance of chromosomes (SMC) complexes shape the genomes of virtually all organisms, but how they function remains incompletely understood. Recent studies in bacteria and eukaryotes have led to a unifying model in which these ring-shaped ATPases act along contiguous DNA segments, processively enlarging DNA loops. In support of this model, single-molecule imaging experiments indicate that Saccharomyces cerevisiae condensin complexes can extrude DNA loops in an ATP-hydrolysis-dependent manner in vitro. Here, using time-resolved high-throughput chromosome conformation capture (Hi-C), we investigate the interplay between ATPase activity of the Bacillus subtilis SMC complex and loop formation in vivo. We show that point mutants in the SMC nucleotide-binding domain that impair but do not eliminate ATPase activity not only exhibit delays in de novo loop formation but also have reduced rates of processive loop enlargement. These data provide in vivo evidence that SMC complexes function as loop extruders.

Keywords: ParB; SMC; TAD; cohesin; condensin; loop extrusion.

Figure 1.. Identification of SMC mutants with decreased ATPase activity.

A, Homology model of the ATPase domain of B. subtilis SMC. Only one of the two composite ATP binding domains is shown. Amino acids that were mutated are indicated (yellow). A schematic of the condensin complex with its head domains (boxed) bound to ATP (blue balls) is shown on the left. B, Growth of wild-type and the relevant mutants in the presence and absence of ParB on agar plates. The 10⁻² and 10⁻⁵ dilutions are shown. Under Hi-C assay conditions (22˚C no IPTG and 37˚C with IPTG in CH medium), the mutants grow similar to wild-type. C, NADH-coupled ATPase activity assay for the indicated mutants. D, Bar graph showing ATPase activities. Error bars show the standard deviation of four replicates. See also Figures S1, S2, Tables S1 and S2.

Figure 2.. SMC ATPase mutants have reduced rates of DNA juxtaposition.

A, Normalized Hi-C interaction maps displaying contact frequencies for pairs of 10 kb bins. The strain (BWX4077) contains wild-type smc, a single parS site at −1˚ and an IPTG-inducible parB allele. Maps show interaction frequencies before and after addition of IPTG. Time after induction is indicated above the maps. Axes present genome positions in degrees and are oriented with the replication origin at the center. The rate of DNA juxtaposition is indicated on the right. See Figure S3 for analysis. The scale bar depicts Hi-C interaction scores for all contact maps presented in this study. B, Hi-C time course of strains containing smc point mutations, K12R (BWX4149), R57A (BWX4078) and F66Y (BWX4152). C, Extent of juxtaposition over time averaged for the left and right arms in the indicated strains. Extrapolation to the abscissa shows the relative lag before juxtaposition begins. See Figure S3 for details. D, Immunoblot analysis of the same strains presented in A and B in the presence of IPTG. The levels of SMC variants, ScpA, ScpB and ParB are similar in all four strains. For the ScpA blot, the bottom band is ScpA and the top band is non-specific. SigA controls for loading. See also Figures S3, S4, S5 and Table S1.