Bürmann, F. | bioRxiv Science

bioRxiv2024

Mechanism of DNA entrapment by the MukBEF SMC complex and its inhibition by a viral DNA mimic

Ring-like structural maintenance of chromosomes (SMC) complexes are crucial for genome organization and operate through mechanisms of DNA entrapment and loop extrusion. Here, we explore the DNA loading process of the bacterial SMC complex MukBEF. Using electron cryomicroscopy (cryo-EM), we demonstrate that ATP binding opens one of MukBEFs three potential DNA entry gates, exposing a DNA capture site that positions DNA at the open neck gate. We discover that the gp5.9 protein of bacteriophage T7 blocks this capture site by DNA mimicry, thereby preventing DNA loading and inactivating MukBEF. We propose a comprehensive and unidirectional loading mechanism in which DNA is first captured at the complexs periphery and then ingested through the DNA entry gate, powered by a single cycle of ATP hydrolysis. These findings illuminate a fundamental aspect of how ubiquitous DNA organizers are primed for genome maintenance and demonstrate how this process can be disrupted by viruses. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=193 HEIGHT=200 SRC="FIGDIR/small/616235v1_ufig1.gif" ALT="Figure 1"> View larger version (68K): org.highwire.dtl.DTLVardef@165a949org.highwire.dtl.DTLVardef@14d1302org.highwire.dtl.DTLVardef@11afb6forg.highwire.dtl.DTLVardef@71694b_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2021

The bacterial actin-like cell division protein FtsA forms curved antiparallel double filaments upon binding of FtsN

Cell growth and division of walled bacteria depend on the synthesis and remodelling of peptidoglycan (PG). These activities are carried out by two multiprotein complexes, the elongasome and the divisome during cell elongation and division, respectively. Filaments of tubulin-like FtsZ form the cytoplasmic scaffold for divisome assembly, the Z-ring. In E. coli, the actin homologue FtsA anchors the Z-ring to the membrane and recruits downstream divisome components, including bitopic FtsN. FtsN is recruited late and activates the periplasmic PG synthase FtsWI. To start unravelling the activation mechanism involving FtsA and FtsN, we showed that E. coli FtsA forms antiparallel double filaments on lipid monolayers when also binding FtsNs cytoplasmic tail, and that Vibrio maritimus FtsA crystallised as an equivalent double filament. We structurally located the FtsA-FtsN interaction site in FtsAs IA-IC interdomain cleft and confirmed FtsA double filament formation in vivo using site-specific cysteine cross-linking. FtsA-FtsN double filaments reconstituted on and in liposomes preferred negative Gaussian curvature, as was previously shown for the elongasomes actin, MreB. MreB filaments serve as curvature-sensing "rudders", orienting insertion of PG around the cells circumference. We propose that curved antiparallel FtsA double filaments function similarly in the divisome: FtsA filaments, together with dynamic FtsZ filaments orient and concentrate cell-constricting septal PG synthesis in the division plane.

microbiology↗