Biology subjects

Kerssemakers, J. W. J.

Publications and source records attributed to Kerssemakers, J. W. J..

6 recordsLinked to original sources

bioRxiv2022

MukBEF-dependent chromosomal organization in widened Escherichia coli

The bacterial chromosome is spatially organized through protein-mediated compaction, supercoiling, and cell-boundary confinement. Structural Maintenance of Chromosomes (SMC) complexes are a major class of chromosome-organizing proteins present throughout all domains of life. Here, we study the role of the Escherichia coli SMC complex MukBEF in chromosome architecture and segregation. Using quantitative live-cell imaging of shape-manipulated cells, we show that MukBEF is crucial to preserve the toroidal topology of the E. coli chromosome and that it is non-uniformly distributed along the chromosome: it prefers locations towards the origin and away from the terminus of replication, and it is unevenly distributed over the origin of replication along the two chromosome arms. Using an ATP hydrolysis-deficient MukB mutant, we find that MukBEF translocation along the chromosome is ATP-dependent, in contrast to its loading onto DNA. MukBEF and MatP are furthermore found to be essential for sister chromosome decatenation. We propose a model that explains how MukBEF, MatP, and their interacting partners organize the chromosome and contribute to sister segregation and recombination. The combination of bacterial cell-shape modification and quantitative fluorescence microscopy paves way to investigating chromosome-organization factors in vivo.

microbiology↗

bioRxiv2020

Resolving the step size in condensin-driven DNA loop extrusion identifies ATP binding as the step-generating process

The condensin SMC protein complex organizes chromosomal structure by extruding loops of DNA. Its ATP-dependent motor mechanism remains unclear but likely involves steps associated with large conformational changes within the ~50 nm protein complex. Here, using high-resolution magnetic tweezers, we resolve single steps in the loop extrusion process by individual yeast condensins. The measured median step sizes range between 20-40 nm at forces of 1.0-0.2 pN, respectively, comparable with the holocomplex size. These large steps show that, strikingly, condensin typically reels in DNA in very sizeable amounts with ~200 bp on average per single extrusion step at low force, and occasionally even much larger, exceeding 500 bp per step. Using Molecular Dynamics simulations, we demonstrate that this is due to the structural flexibility of the DNA polymer at these low forces. Using ATP-binding-impaired and ATP-hydrolysis-deficient mutants, we find that ATP binding is the primary step-generating stage underlying DNA loop extrusion. We discuss our findings in terms of a scrunching model where a stepwise DNA loop extrusion is generated by an ATP-binding-induced engagement of the hinge and the globular domain of the SMC complex.

biophysics↗

bioRxiv2020

Bulk-surface coupling reconciles Min-protein pattern formation in vitro and in vivo

Self-organisation of Min proteins is responsible for the spatial control of cell division in Escherichia coli, and has been studied both in vivo and in vitro. Intriguingly, the protein patterns observed in these settings differ qualitatively and quantitatively. This puzzling dichotomy has not been resolved to date. Using reconstituted proteins in laterally wide microchambers with a well-controlled height, we show that the Min protein dynamics on the membrane crucially depend on bulk gradients normal to the membrane. A theoretical analysis shows that in vitro patterns at low bulk height are driven by the same lateral oscillation mode as pole-to-pole oscillations in vivo. At larger bulk height, additional vertical oscillation modes set in, marking the transition to a qualitatively different in vitro regime. Our work qualitatively resolves the Min systems in vivo/in vitro conundrum and provides important insights on the mechanisms underlying protein patterns in bulk-surface coupled systems.

biophysics↗

bioRxiv2019

Direct observation of independently moving replisomes in Escherichia coli

The replication and transfer of genomic material from a cell to its progeny are vital processes in all living systems. Here we visualize the process of chromosome replication in E. coli cells with an increased width. Monitoring the replication of single chromosomes yields clear examples of replication bubbles that reveal that the two replisomes move independently from the origin to the terminus of replication along each of the two arms of the circular chromosome, providing direct support for the so-called train-track model, and against a factory model for replisomes. The origin of replication duplicates near midcell, initially splitting to random directions and subsequently towards the poles. The probability of successful segregation of chromosomes significantly decreases with increasing cell width, indicating that chromosome confinement by the cell boundary is an important driver of DNA segregation. Our findings resolve long standing questions in bacterial chromosome organization.

cell biology↗

bioRxiv2019

DNA-loop extruding condensin complexes can traverse one another

Condensin, a key member of the Structure Maintenance of Chromosome (SMC) protein complexes, has recently been shown to be a motor that extrudes loops of DNA1. It remains unclear, however, how condensin complexes work together to collectively package DNA into the chromosomal architecture. Here, we use time-lapse single-molecule visualization to study mutual interactions between two DNA-loop-extruding yeast condensins. We find that these one-side-pulling motor proteins are able to dynamically change each others DNA loop sizes, even when located large distances apart. When coming into close proximity upon forming a loop within a loop, condensin complexes are, surprisingly, able to traverse each other and form a new type of loop structure, which we term Z loop - three double-stranded DNA helices aligned in parallel with one condensin at each edge. These Z-loops can fill gaps left by single loops and can form symmetric dimer motors that reel in DNA from both sides. These new findings indicate that condensin may achieve chromosomal compaction using a variety of looping structures.

biophysics↗

bioRxiv2018

Direct Imaging of the circular chromosome of a live bacterium

New assays for quantitative imaging1-6 and sequencing7-11 have yielded great progress towards understanding the organizational principles of chromosomes. Yet, even for the well-studied model bacterium Escherichia coli, many basic questions remain unresolved regarding chromosomal (sub-)structure2,11, its mechanics1,2,12 and dynamics13,14, and the link between structure and function1,15,16. Here we resolve the spatial organization of the circular chromosome of bacteria by directly imaging the chromosome in live E. coli cells with a broadened cell shape. The chromosome was observed to exhibit a torus topology with a 4.2 m toroidal length and 0.4 m bundle thickness. On average, the DNA density along the chromosome shows dense right and left arms that branch from a lower-density origin of replication, and are connected at the terminus of replication by an ultrathin flexible string of DNA. At the single-cell level, the DNA density along the torus is found to be strikingly heterogeneous, with blob-like Mbp-size domains that undergo major dynamic rearrangements, splitting and merging at a minute timescale. We show that prominent domain boundaries at the terminus and origin of replication are induced by MatP proteins, while weaker transient domain boundaries are facilitated by the global transcription regulators HU and Fis. These findings provide an architectural basis for the understanding of the spatial organization of bacterial genomes.

microbiology↗