In eukaryotes, SMC complexes form TADs by extruding DNA loops and being stalled by roadblock proteins. It remains unclear whether a similar mechanism of domain formation exists in prokaryotes. Using 3C-seq, we show that an archaeal homolog of the bacterial Smc-ScpAB complex organizes the genome of Thermococcus kodakarensis into TAD-like domains. We find that TrmBL2, a DNA-binding protein that forms a stiff nucleoprotein filament, stalls the T. kodakarensis SMC complex and establishes a boundary at the site-specific recombination site dif. TrmBL2 stalls the SMC complex at tens of additional non-boundary loci with lower efficiency. Intriguingly, the stalling efficiency is correlated with structural properties of underlying DNA sequences. Our study illuminates a eukaryotic-like mechanism of domain formation in archaea and a role of intrinsic DNA structure in large-scale genome organization.

Cells were crosslinked, permeabilized, and treated with AluI and HaeIII to digest genomic DNA. The insoluble fraction was collected and used for proximity ligation. After reversal of crosslinks, the DNA was extracted with phenol:chloroform:isoamyl alcohol, precipitated in ethanol, and sonicated.