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Bacillus subtilis ParB forms multimeric networks involving non-specific DNA binding leading to DNA condensation. Previously, we found that an excess of the free C-terminal domain (CTD) of ParB impeded DNA condensation or promoted decondensation of pre-assembled networks (Fisher et al., 2017). However, interpretation of the molecular basis for this phenomenon was complicated by our inability to uncouple protein binding from DNA condensation. Here, we have combined lateral magnetic tweezers with TIRF microscopy to simultaneously control the restrictive force against condensation and to visualise ParB protein binding by fluorescence. At non-permissive forces for condensation, ParB binds non-specifically and highly dynamically to DNA. Our new approach concluded that the free CTD blocks the formation of ParB networks by heterodimerisation with full length DNA-bound ParB. This strongly supports a model in which the CTD acts as a key bridging interface between distal DNA binding loci within ParB networks., European Research Council (ERC) (Grant agreement no. 681299). Basque Country Government Department of Education, Language Policy and Culture.(ref.PRE_2013_11_1174). Biotechnology and Biological Sciences Research Council. (1363883). Wellcome Trust (100401 and 077368).

Summary Eukaryotic SMC complexes, cohesin, condensin, and Smc5/6, use ATP hydrolysis to power a plethora of functions requiring organization and restructuring of eukaryotic chromosomes in interphase and during mitosis. The Smc5/6 mechanism of action and its activity on DNA are largely unknown. Here we purified the budding yeast Smc5/6 holocomplex and characterized its core biochemical and biophysical activities. Purified Smc5/6 exhibits DNA-dependent ATP hydrolysis and SUMO E3 ligase activity. We show that Smc5/6 binds DNA topologically with affinity for supercoiled and catenated DNA templates. Employing single-molecule assays to analyze the functional and dynamic characteristics of Smc5/6 bound to DNA, we show that Smc5/6 locks DNA plectonemes and can compact DNA in an ATP-dependent manner. These results demonstrate that the Smc5/6 complex recognizes DNA tertiary structures involving juxtaposed helices and might modulate DNA topology by plectoneme stabilization and local compaction., Graphical Abstract, Highlights • Purification of enzymatically active Smc5/6 yeast holocomplex • Smc5/6 coiled-coils exhibit a folded conformation • Smc5/6 stabilizes DNA plectonemes • Smc5/6 compacts DNA against low forces in an ATP-dependent manner, Gutierrez-Escribano et al. purify the entire Smc5/6 holocomplex, retaining full enzymatic function. The Smc5/6 complex compacts DNA against low force and stabilizes DNA crosses present in supercoiled and catenated DNA. These findings indicate that many Smc5/6 functions occur through stabilization of DNA tertiary structure.

Combining single-molecule techniques with fluorescence microscopy has attracted much interest because it allows the correlation of mechanical measurements with directly visualized DNA:protein interactions. In particular, combination with total internal reflection fluorescence microscopy (TIRF) is advantageous because of the high signal-to-noise ratio this technique achieves. This, however, requires stretching long DNA molecules across the surface of the flow cell to maximize polymer exposure to the excitation light. In this work, we develop a module to laterally stretch DNA molecules at a constant force, which can be easily implemented in regular or combined magnetic tweezers (MT)-TIRF setups. The pulling module is further characterized in standard flow cells of different thicknesses and glass capillaries, using two types of micrometer size superparamagnetic beads, long DNA molecules, and a home-built device to rotate capillaries with mrad precision. The force range achieved by the magnetic pulling module was between 0.1 and 30 pN. A formalism for estimating forces in flow-stretched tethered beads is also proposed, and the results compared with those of lateral MT, demonstrating that lateral MT achieve higher forces with lower dispersion. Finally, we show the compatibility with TIRF microscopy and the parallelization of measurements by characterizing DNA binding by the centromere-binding protein ParB from Bacillus subtilis. Simultaneous MT pulling and fluorescence imaging demonstrate the non-specific binding of BsParB on DNA under conditions restrictive to condensation., We thank the financial support from the Spanish MINECO (FIS2014-58328-P) and from (ERC) under the European Union’s Horizon2020 Research and Innovation programme (grant agreement No 681299). J. M. M. acknowledges a Predoctoral PhD fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174). ). G. L. M. F was supported by the Biotechnology and Biological Sciences Research Council (1363883). M. S. D was supported by the Wellcome Trust (100401 and 077368).

ADP-glucose pyrophosphorylase (AGPase) catalyzes the rate-limiting step of bacterial glycogen and plant starch biosynthesis, the most common carbon storage polysaccharides in nature. A major challenge is to understand how AGPase activity is regulated by metabolites in the energetic flux within the cell. Here we report crystal structures of the homotetrameric AGPase from Escherichia coli in complex with its physiological positive and negative allosteric regulators, fructose-1,6-bisphosphate (FBP) and AMP, and sucrose in the active site. FBP and AMP bind to partially overlapping sites located in a deep cleft between glycosyltransferase A-like and left-handed β helix domains of neighboring protomers, accounting for the fact that sensitivity to inhibition by AMP is modulated by the concentration of the activator FBP. We propose a model in which the energy reporters regulate EcAGPase catalytic activity by intra-protomer interactions and inter-protomer crosstalk, with a sensory motif and two regulatory loops playing a prominent role.

Single-molecule experiments usually take place in flow cells. This experimental approach is essential for experiments requiring a liquid environment, but is also useful to allow the exchange of reagents before or during measurements. This is crucial in experiments that need to be triggered by ligands or require a sequential addition of proteins. Home-fabricated flow cells using two glass coverslips and a gasket made of paraffin wax are a widespread approach. The volume of the flow cell can be controlled by modifying the dimensions of the channel while the reagents are introduced using a syringe pump. In this system, high flow rates disturb the biological system, whereas lower flow rates lead to the generation of a reagent gradient in the flow cell. For very precise measurements it is thus desirable to have a very fast exchange of reagents with minimal diffusion. We propose the implementation of multistream laminar microfluidic cells with two inlets and one outlet, which achieve a minimum fluid switching time of 0.25 s. We additionally define a phenomenological expression to predict the boundary switching time for a particular flow cell cross section. Finally, we study the potential applicability of the platform to study kinetics at the single molecule level., European Research Council (ERC) under the European Union Horizon 2020 research and innovation (grant agreement No 681299). The work in the Moreno-Herrero laboratory was also supported by a Spanish Ministry of Economy and Competitiveness Grant, BFU2017-83794-P (AEI/FEDER, UE) to F.M.-H., and Comunidad de Madrid Grants, Tec4Bio – S2018/NMT-4443 and NanoBioCancer – Y2018/BIO-4747 to F.M.-H. J.M.-M. acknowledges support for pre-doctoral fellowship from the Basque Country Government Department of Education, Language Policy and Culture (ref. PRE_2013_11_1174).