Filament Depolymerization Can Explain Chromosome Pulling during Bacterial Mitosis

Chromosome segregation is fundamental to all cells, but the force-generating mechanisms underlying chromosome translocation in bacteria remain mysterious. Caulobacter crescentus utilizes a depolymerization-driven process in which a ParA protein structure elongates from the new cell pole, binds to a ParB-decorated chromosome, and then retracts via disassembly, pulling the chromosome across the cell. This poses the question of how a depolymerizing structure can robustly pull the chromosome that disassembles it. We perform Brownian dynamics simulations with a simple, physically consistent model of the ParABS system. The simulations suggest that the mechanism of translocation is “self-diffusiophoretic”: by disassembling ParA, ParB generates a ParA concentration gradient so that the ParA concentration is higher in front of the chromosome than behind it. Since the chromosome is attracted to ParA via ParB, it moves up the ParA gradient and across the cell. We find that translocation is most robust when ParB binds side-on to ParA filaments. In this case, robust translocation occurs over a wide parameter range and is controlled by a single dimensionless quantity: the product of the rate of ParA disassembly and a characteristic relaxation time of the chromosome. This time scale measures the time it takes for the chromosome to recover its average shape after it is has been pulled. Our results suggest explanations for observed phenomena such as segregation failure, filament-length-dependent translocation velocity, and chromosomal compaction

A Defined Terminal Region of the E. coli Chromosome Shows Late Segregation and High FtsK Activity

Background: The FtsK DNA-translocase controls the last steps of chromosome segregation in E. coli. It translocates sister chromosomes using the KOPS DNA motifs to orient its activity, and controls the resolution of dimeric forms of sister chromosomes by XerCD-mediated recombination at the dif site and their decatenation by TopoIV. Methodology: We have used XerCD/dif recombination as a genetic trap to probe the interaction of FtsK with loci located in different regions of the chromosome. This assay revealed that the activity of FtsK is restricted to a,400 kb terminal region of the chromosome around the natural position of the dif site. Preferential interaction with this region required the tethering of FtsK to the division septum via its N-terminal domain as well as its translocation activity. However, the KOPSrecognition activity of FtsK was not required. Displacement of replication termination outside the FtsK high activity region had no effect on FtsK activity and deletion of a part of this region was not compensated by its extension to neighbouring regions. By observing the fate of fluorescent-tagged loci of the ter region, we found that segregation of the FtsK high activity region is delayed compared to that of its adjacent regions. Significance: Our results show that a restricted terminal region of the chromosome is specifically dedicated to the last step

Direct observation of DNA threading in flap endonuclease complexes

Maintenance of genome integrity requires that branched nucleic acid molecules are accurately processed to produce double-helical DNA. Flap endonucleases are essential enzymes that trim such branched molecules generated by Okazaki fragment synthesis during replication. Here, we report crystal structures of bacteriophage T5 flap endonuclease in complexes with intact DNA substrates, and products, at resolutions of 1.9–2.2 Å. They reveal single-stranded DNA threading through a hole in the enzyme enclosed by an inverted Vshaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate juxtaposing the scissile phosphate and essential catalytic metal ions. A series of complexes and biochemical analyses show how the substrate’s single-stranded branch approaches, threads through, and finally emerges on the far side of the enzyme. Our studies suggest that substrate recognition involves an unusual “flycasting, thread, bend and barb” mechanis

Single-molecule imaging of DNA curtains reveals mechanisms of KOPS sequence targeting by the DNA translocase FtsK

FtsK is a hexameric DNA translocase that participates in the final stages of bacterial chromosome segregation. Here we investigate the DNA-binding and translocation activities of FtsK in real time by imaging fluorescently tagged proteins on nanofabricated curtains of DNA. We show that FtsK preferentially loads at 8-bp KOPS (FtsK Orienting Polar Sequences) sites and that loading is enhanced in the presence of ADP. We also demonstrate that FtsK locates KOPS through a mechanism that does not involve extensive 1D diffusion at the scale of our resolution. Upon addition of ATP, KOPS-bound FtsK translocates in the direction dictated by KOPS polarity, and once FtsK has begun translocating it does not rerecognize KOPS from either direction. However, FtsK can abruptly change directions while translocating along DNA independent of KOPS, suggesting that the ability to reorient on DNA does not arise from DNA sequence-specific effects. Taken together, our data support a model in which FtsK locates KOPS through random collisions, preferentially engages KOPS in the ADP-bound state, translocates in the direction dictated by the polar orientation of KOPS, and is incapable of recognizing KOPS while translocating along DNAclos

Sequence-directed DNA translocation by purified FtsK.

DNA translocases are molecular motors that move rapidly along DNA using adenosine triphosphate as the source of energy. We directly observed the movement of purified FtsK, an Escherichia coli translocase, on single DNA molecules. The protein moves at 5 kilobases per second and against forces up to 60 piconewtons, and locally reverses direction without dissociation. On three natural substrates, independent of its initial binding position, FtsK efficiently translocates over long distances to the terminal region of the E. coli chromosome, as it does in vivo. Our results imply that FtsK is a bidirectional motor that changes direction in response to short, asymmetric directing DNA sequences