53 research outputs found
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 âself-diffusiophoreticâ: 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
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 1.9â2.2 Ă
. 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
substrateâs 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 âflycasting,
thread, bend and barbâ mechanis
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
- âŚ