Cryo-EM structure of MukBEF reveals DNA loop entrapment at chromosomal unloading sites

Abstract

Summary The ring-like structural maintenance of chromosomes (SMC) complex MukBEF folds the genome of Escherichia coli and related bacteria into large loops, presumably by active DNA loop extrusion. MukBEF activity within the replication terminus macrodomain is suppressed by the sequence-specific unloader MatP. Here, we present the complete atomic structure of MukBEF in complex with MatP and DNA as determined by electron cryomicroscopy (cryo-EM). The complex binds two distinct DNA double helices corresponding to the arms of a plectonemic loop. MatP-bound DNA threads through the MukBEF ring, while the second DNA is clamped by the kleisin MukF, MukE, and the MukB ATPase heads. Combinatorial cysteine cross-linking confirms this topology of DNA loop entrapment in vivo . Our findings illuminate how a class of near-ubiquitous DNA organizers with important roles in genome maintenance interacts with the bacterial chromosome.

The ParB clamp docks onto Smc for DNA loading via a joint-ParB interface

“…The symmetric nature of the bacterial Smc dimer (Figure 7A) allows for two joint-ParB interfaces on the asymmetric Smc-ScpAB holo-complex. ParB may either bind on the same side as ScpAB ('front'), which would likely result in a steric clash since the middle part of ScpAB occupies a similar area on top of ATP-engaged Smc heads (Figure 7A) (according to the position of the corresponding kite subunits in DNA-clamping MukBEF) (Bürmann et al, 2021). Alternatively, ParB may approach from the other side ('back') and thus avoid a steric clash.…”

“…The AcpP protein has an important stimulatory effect on the ATPase activity of MukBEF (Josh et al, 2021). Both proteins bind to the joint in MukBEF (the two joints actually) (Bürmann et al, 2021). The hawk subunit Scc2 is a loading as well as processivity factor for DNA loop extrusion by cohesin (Davidson et al, 2019).…”

“…To understand how closely these joint interfaces might be related to one another, we superimposed the joint domain in these structures. Superimposition with MukB (PDB: 7NZ3) is challenging due to the significantly diverged structure of the joint (Bürmann et al, 2021). The Smc3 joint (PDB: 6YUF) superimposed significantly better with the Smc joint and intriguingly showed that the hawk-joint interface overlaps well with the joint-ParB interface (Figure 6D) (Higashi et al, 2020).…”

DNA tension-modulated translocation and loop extrusion by SMC complexes revealed by molecular dynamics simulations

“…A structural feature that varies among different SMCC species is the degree of folding of the coiled-coils at the elbow sites, to bring the hinge domain towards the kleisin region (e.g., Figure 1A , yeast condensin). Yeast condensin ( 20 ), cohesin ( 26 , 27 ) and E. coli MukBEF ( 26 , 28 ) are known to have this folding, while SMC5/6 ( 29 , 30 ) and B. subtilis bsSMC ( 31 ) are thought not to have it. As will be discussed, our model is not strongly dependent on the precise geometry of the complex, other than having the coiled-coils adjacent and the upper compartment ‘closed’ in the apo state, and having the upper compartment open when ATP is bound.…”

Single-Molecule/Cell Analyses Reveal Principles of Genome-Folding Mechanisms in the Three Domains of Life

“…Single-molecule tracking of B. subtilis nucleoids revealed that SMC operates by different patterns of motion compared to other DNA-condensing NAPs such as gyrase and HBsu (an HU family protein) [85,86]. Electron cryomicroscopy (cryo-EM) single-particle analysis revealed the detailed mechanism of how E. coli MukBEF entraps two distinct DNA strands when bound to the unloader MatP [87].…”