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Abstract: DNA transformation is a widespread process allowing bacteria to capture free DNA by using filamentous nano-machines composed of type IV pilins. These proteins can act as DNA receptors as demonstrated by the finding that Neisseria meningitidis ComP minor pilin has intrinsic DNA-binding ability. ComP binds DNA better when it contains the DNA-uptake sequence (DUS) motif abundant in this species genome, playing a role in its trademark ability to selectively take up its own DNA. Here, we report high-resolution structures for meningococcal ComP and Neisseria subflava ComPsub, which recognize different DUS motifs. We show that they are structurally identical type IV pilins that pack readily into filament models and display a unique DD region delimited by two disulfide bonds. Functional analysis of ComPsub defines a new mode of DNA binding involving the DD region, adapted for exported DNA receptors.

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Abstract: Purpose: We aimed to characterize alterations in the posterior scleral collagen microstructure before detectable disease onset in a canine model of open-angle glaucoma caused by an ADAMTS10 mutation. Methods: Collagen orientation, anisotropy degree (proportion of preferentially aligned collagen), and relative density were measured at 0.4 mm spatial resolution using synchrotron wide-angle X-ray scattering. For statistical evaluation of structure parameters, regional averages of the peripapillary and mid-posterior sclera were compared between ADAMTS10 mutant (affected) dogs (n = 3) and age-matched (carrier) controls (n = 3). Results: No marked differences in the general pattern of preferential collagen fibril orientation were noted between the control and affected dogs. The peripapillary sclera of all specimens featured strongly aligned circumferential collagen ringing the optic nerve head. Collagen anisotropy was significantly reduced in the mid-posterior sclera of the affected dogs (carrier: 0.27 +/- 0.11; affected: 0.24 +/- 0.10; p = 0.032) but was not statistically significantly different in the peripapillary sclera (carrier: 0.46 +/- 0.15; affected: 0.45 +/- 0.17; p = 0.68). Collagen density was statistically significantly reduced in the affected dogs for the mid-posterior sclera (carrier: 28.1 +/- 9.14; affected: 18.3 +/- 5.12; p< 0.0001) and the peripapillary sclera (carrier: 34.6 +/- 9.34; affected: 21.1 +/- 6.97; p = 0.0002). Conclusions: Significant alterations in the posterior scleral collagen microstructure are present before the onset of clinical glaucoma in ADAMTS10 mutant dogs. A reduction in fibrous collagen density is likely an important contributory factor in the previously reported mechanical weakening of the sclera in this model. Baseline scleral abnormalities have the potential to interact with intraocular pressure (IOP) elevations in determining the course of glaucoma progression in animal models of the disease, and potentially in human glaucoma.

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Abstract: The breakdown of plant cell wall (PCW) glycans is an important biological and industrial process. Noncatalytic carbohydrate binding modules (CBMs) fulfill a critical targeting function in PCW depolymerization. Defining the portfolio of CBMs, the CBMome, of a PCW degrading system is central to understanding the mechanisms by which microbes depolymerize their target substrates. Ruminococcus flavefaciens, a major PCW degrading bacterium, assembles its catalytic apparatus into a large multienzyme complex, the cellulosome. Significantly, bioinformatic analyses of the R. flavefaciens cellulosome failed to identify a CBM predicted to bind to crystalline cellulose, a key feature of the CBMome of other PCW degrading systems. Here, high throughput screening of 177 protein modules of unknown function was used to determine the complete CBMome of R. flavefaciens. The data identified six previously unidentified CBMfamilies that targeted beta-glucans, beta-mannans, and the pectic polysaccharide homogalacturonan. The crystal structures of four CBMs, in conjunction with site-directed mutagenesis, provide insight into the mechanism of ligand recognition. In the CBMs that recognize beta-glucans and beta-mannans, differences in the conformation of conserved aromatic residues had a significant impact on the topology of the ligand binding cleft and thus ligand specificity. A cluster of basic residues in CBM77 confers calcium-independent recognition of homogalacturonan, indicating that the carboxylates of galacturonic acid are key specificity determinants. This report shows that the extended repertoire of proteins in the cellulosome of R. flavefaciens contributes to an extended CBMome that supports efficient PCW degradation in the absence of CBMs that specifically target crystalline cellulose.

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Abstract: Replisome assembly at eukaryotic replication forks connects the DNA helicase to DNA polymerases and many other factors. The helicase binds the leading-strand polymerase directly, but is connected to the Pol alpha lagging-strand polymerase by the trimeric adaptor Ctf4. Here, we identify new Ctf4 partners inaddition to Pol alpha and helicase, all of which contain a "Ctf4-interacting-peptide" or CIP-box. Crystallographic analysis classifies CIP-boxes into two related groups that target different sites on Ctf4. Mutations in the CIP-box motifs of the Dna2 nuclease or the rDNA-associated protein Tof2 do not perturb DNA synthesis genome-wide, but instead lead to a dramatic shortening of chromosome 12 that contains the large array of rDNA repeats. Our data reveal unexpected complexity of Ctf4 function, as a hub that connects multiple accessory factors to the replisome. Most strikingly, Ctf4-dependent recruitment of CIP-box proteins couples other processes to DNA synthesis, including rDNA copy-number regulation.

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Abstract: Maintenance of genome integrity requires that branched nucleic acid molecules be 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, which is enclosed by an inverted V-shaped helical arch straddling the active site. Residues lining the hole induce an unusual barb-like conformation in the DNA substrate, thereby 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 'fly-casting, thread, bend and barb' mechanism.