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

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

Maine Summer is Back: Your Regional Guide

Extended article detailing summer activity options from Southern Maine to Northern Maine

The Ultimate Maine Summer Guide

An overview of the regional activities and delicacies which make a summer in Maine special. A section for each region of the state - Down East and Mount Desert Island, Southern Maine, Central and Western Maine, Greater Portland, Midcoast, and Northern Maine - is broken down into categories - The Fest, The Treat, The Feat, The Trail, The Beach, The Show, and The Chow - which briefly detail special things to do, places to visit, and food to enjoy in each area. Entries run the gamut from Biking the carriage roads of Acadia National Park, to farm-to-bus crepes from the electric-green YumBus at farmers\u27 markets in Bangor and Orono

SpoIIIE strips proteins off the DNA during chromosome translocation

The FtsK/SpoIIIE family of DNA transporters are responsible for translocating missegregated chromosomes after the completion of cell division. An extreme example of this post-cytokinetic DNA segregation occurs during spore formation in the bacterium Bacillus subtilis, where SpoIIIE pumps three-quarters of the chromosome (>3 megabases) into one of the two daughter cells. Here, we investigate the fate of the proteins associated with the translocated DNA. Taking advantage of several unique features of Bacillus sporulation, we demonstrate that RNA polymerase, transcription factors, and chromosome remodeling proteins are stripped off the DNA during translocation of the chromosome into the forespore compartment. Furthermore, we show that in vitro the soluble ATPase domain of SpoIIIE can displace RNA polymerase bound to DNA, suggesting that SpoIIIE alone is capable of this wire-stripping activity. Our data suggest that the bulk of the forespore chromosome is translocated naked into the forespore compartment. We propose that the translocation-stripping activity of SpoIIIE plays a key role in reprogramming developmental gene expression in the forespore