Defects in the GINS complex increase the instability of repetitive sequences via a recombination-dependent mechanism

Faithful replication and repair of DNA lesions ensure genome maintenance. During replication in eukaryotic cells, DNA is unwound by the CMG helicase complex, which is composed of three major components: the Cdc45 protein, Mcm2-7, and the GINS complex. The CMG in complex with DNA polymerase epsilon (CMG-E) participates in the establishment and progression of the replisome. Impaired functioning of the CMG-E was shown to induce genomic instability and promote the development of various diseases. Therefore, CMG-E components play important roles as caretakers of the genome. In Saccharomyces cerevisiae, the GINS complex is composed of the Psf1, Psf2, Psf3, and Sld5 essential subunits. The Psf1-1 mutant form fails to interact with Psf3, resulting in impaired replisome assembly and chromosome replication. Here, we show increased instability of repeat tracts (mononucleotide, dinucleotide, trinucleotide and longer) in yeast psf1-1 mutants. To identify the mechanisms underlying this effect, we analyzed repeated sequence instability using derivatives of psf1-1 strains lacking genes involved in translesion synthesis, recombination, or mismatch repair. Among these derivatives, deletion of RAD52, RAD51, MMS2, POL32, or PIF1 significantly decreased DNA repeat instability. These results, together with the observed increased amounts of single-stranded DNA regions and Rfa1 foci suggest that recombinational mechanisms make important contributions to repeat tract instability in psf1-1 cells. We propose that defective functioning of the CMG-E complex in psf1-1 cells impairs the progression of DNA replication what increases the contribution of repair mechanisms such as template switch and break-induced replication. These processes require sequence homology search which in case of a repeated DNA tract may result in misalignment leading to its expansion or contraction

Increased contribution of DNA polymerase delta to the leading strand replication in yeast with an impaired CMG helicase complex

DNA replication is performed by replisome proteins, which are highly conserved from yeast to humans. The CMG [Cdc45-Mcm2–7-GINS(Psf1–3, Sld5)] helicase unwinds the double helix to separate the leading and lagging DNA strands, which are replicated by the specialized DNA polymerases epsilon (Pol ε) and delta (Pol δ), respectively. This division of labor was confirmed by both genetic analyses and in vitro studies. Exceptions from this rule were described mainly in cells with impaired catalytic polymerase ε subunit. The central role in the recruitment and establishment of Pol ε on the leading strand is played by the CMG complex assembled on DNA during replication initiation. In this work we analyzed the consequences of impaired functioning of the CMG complex for the di�vision labor between DNA polymerases on the two replicating strands. We showed in vitro that the GINSPsf1–1 complex poorly bound the Psf3 subunit. In vivo, we observed increased rates of L612M Pol δ-specific mutations during replication of the leading DNA strand in psf1–1 cells. These findings indicated that defective functioning of GINS impaired leading strand replication by Pol ε and necessitated involvement of Pol δ in the synthesis on this strand with a possible impact on the distribution of mutations and genomic stability. These are the first results to imply that the division of labor between the two main replicases can be severely influenced by a defective nonpolymerase subunit of the replisome

A Unique Mechanochemical Redox Reaction Yielding Nanostructured Double Perovskite Sr2_{2}FeMoO6_{6} With an Extraordinarily High Degree of Anti-Site Disorder

Strontium ferromolybdate, Sr(2)FeMoO(6), is an important member of the family of double perovskites with the possible technological applications in the field of spintronics and solid oxide fuel cells. Its preparation via a multi-step ceramic route or various wet chemistry-based routes is notoriously difficult. The present work demonstrates that Sr(2)FeMoO(6) can be mechanosynthesized at ambient temperature in air directly from its precursors (SrO, α-Fe, MoO(3)) in the form of nanostructured powders, without the need for solvents and/or calcination under controlled oxygen fugacity. The mechanically induced evolution of the Sr(2)FeMoO(6) phase and the far-from-equilibrium structural state of the reaction product are systematically monitored with XRD and a variety of spectroscopic techniques including Raman spectroscopy, (57)Fe Mössbauer spectroscopy, and X-ray photoelectron spectroscopy. The unique extensive oxidation of iron species (Fe(0) → Fe(3+)) with simultaneous reduction of Mo cations (Mo(6+) → Mo(5+)), occuring during the mechanosynthesis of Sr(2)FeMoO(6), is attributed to the mechanically triggered formation of tiny metallic iron nanoparticles in superparamagnetic state with a large reaction surface and a high oxidation affinity, whose steady presence in the reaction mixture of the milled educts initiates/promotes the swift redox reaction. High-resolution transmission electron microscopy observations reveal that the mechanosynthesized Sr(2)FeMoO(6), even after its moderate thermal treatment at 923 K for 30 min in air, exhibits the nanostructured nature with the average particle size of 21(4) nm. At the short-range scale, the nanostructure of the as-prepared Sr(2)FeMoO(6) is characterized by both, the strongly distorted geometry of the constituent FeO(6) octahedra and the extraordinarily high degree of anti-site disorder. The degree of anti-site disorder ASD = 0.5, derived independently from the present experimental XRD, Mössbauer, and SQUID magnetization data, corresponds to the completely random distribution of Fe(3+) and Mo(5+) cations over the sites of octahedral coordination provided by the double perovskite structure. Moreover, the fully anti-site disordered Sr(2)FeMoO(6) nanoparticles exhibit superparamagnetism with the blocking temperature T (B) = 240 K and the deteriorated effective magnetic moment μ = 0.055 μ (B) per formula unit

Estimating Pore-Size Distributions of Moderately Hydrophobic Mesoporous Solids

In this paper, standard reduced data for adsorption of nitrogen on moderately hydrophobic surface are reported. This surface was obtained by chemical modification of macroporous silica with 3-mercaptopropyltrimethoxysilane. In addition, the statistical film thickness, t curve, is derived using these data and nitrogen adsorption isotherms measured on two large-pore ordered mesoporous silica MCM-41 samples modified with the same organosilane. The application of these reference adsorption data is shown in the evaluation of pore-size distribution and in the related characterization of mesoporous materials with moderate surface hydrophobicity

Versatile Surfactant/Swelling-Agent Template for Synthesis of Large-Pore Ordered Mesoporous Silicas and Related Hollow Nanoparticles

A surfactant/swelling-agent pair suitable for templating a variety of well-defined large-pore nanoporous silicas was identified. The pair includes a poly­(ethylene oxide)-poly­(propylene oxide)-poly­(ethylene oxide), PEO-PPO-PEO, block copolymer surfactant (Pluronic F127, EO₁₀₆PO₇₀EO₁₀₆) with a large fraction of long hydrophilic PEO blocks and a swelling agent (toluene) that strongly solubilizes in micelles of the PEO-PPO-PEO surfactant family. Such a combination affords micellar templates for both spherical and cylindrical mesopores with potential to hinder cross-linking of micelle-templated nanostructures due to stabilization of nanoparticles by long PEO chains. Under low-temperature conditions (11–12 °C), the Pluronic F127/toluene pair affords ultralarge-pore FDU-12 (ULP-FDU-12) silica with face-centered cubic structure of spherical mesopores and related hollow nanospheres, as well as large-pore SBA-15 (LP-SBA-15) with two-dimensional hexagonal structure of cylindrical mesopores and related silica nanotubes. ULP-FDU-12 reaches the unit-cell parameter of 69 nm, which is very large. LP-SBA-15 has a unit-cell parameter up to 26 nm and pore diameter up to ∼20 nm and is exceptionally well ordered. The hollow nanospheres and nanotubes are attainable through lowering of the silica-precursor/surfactant ratio. The materials templated by spherical micelles form when the surfactant/swelling-agent solution is kept under stirring for extended periods of time before the addition of the silica precursor. The sizes of entrances to the hollow nanospheres can be continuously tuned by adjusting the hydrothermal treatment temperature. The ordered mesoporous silicas can be converted from open-pore to closed-pore materials through the thermally induced pore closing. The diversity in morphology, pore size, and pore connectivity makes the proposed surfactant/swelling-agent templating system unprecedented in the large mesopore domain

Family of Single-Micelle-Templated Organosilica Hollow Nanospheres and Nanotubes Synthesized through Adjustment of Organosilica/Surfactant Ratio

A family of hollow organosilica nanospheres and nanotubes was synthesized at appropriately low organosilica-precursor/block-copolymer-surfactant ratios. In Pluronic F127 (EO₁₀₆PO₇₀EO₁₀₆) block copolymer templated synthesis of ethylene-bridged organosilicas in the presence of a swelling agent, the lowering of the organosilica-precursor/surfactant ratio led to a change from highly ordered face-centered cubic structure of spherical mesopores to individual hollow spherical nanoparticles. It was hypothesized that at low ratios of organosilica precursor to PEO-PPO-PEO, the framework precursor is solubilized in the micelles and its concentration on their surface is not sufficient to induce appreciable cross-linking between the resulting nanoobjects and the consolidation into larger particles. The inner pore size of the nanospheres was adjusted by varying the micelle expander, allowing us to obtain pore diameters up to ∼20 nm. By employing low precursor/surfactant ratios, hollow spheres of methylene-, ethenylene-, and phenylene-bridged organosilicas were synthesized. Hollow silica spheres were also obtained through judicious choice of block copolymer. The synthesis strategy involving the adjustment of the framework-precursor/surfactant ratio was further extended on organosilica nanotubes synthesized using Pluronic P123 surfactant and cyclohexane as a swelling agent. One can envision a large number of framework compositions for which hollow nanospheres and nanotubes can be obtained using our synthesis approach

Surfactant-Templated Synthesis of Ordered Silicas with Closed Cylindrical Mesopores

Ordered mesoporous silicas with 2-dimensional hexagonal arrays of closed cylindrical pores were synthesized via templating with block copolymer surfactant followed by calcination at appropriately high temperatures. Precursors to closed-pore silicas, including SBA-15 silicas and organosilicas, were selected based on the existence of narrow passages to the mesopores. The increase in calcination temperature to 800–950 °C led to a dramatic decrease in nitrogen uptake by the materials, indicating the loss of accessible mesopores, whereas small-angle X-ray scattering (SAXS) indicated no major structural changes other than the framework shrinkage. Since SAXS patterns for ordered mesoporous materials are related to periodic arrays of mesopores, the existence of closed mesopores was evident, as additionally confirmed by TEM. The formation of closed-pore silicas was demonstrated for ultralarge-pore SBA-15 and large-pore phenylene-bridged periodic mesoporous organosilicas. The increase in the amount of tetraethyl orthosilicate in standard SBA-15 synthesis also allowed us to observe the thermally induced pore closing. It is hypothesized that the presence of porous plugs in the cylindrical mesopores and/or caps at their ends was responsible for the propensity to the pore closing at sufficiently high temperatures. The observed behavior is likely to be relevant to a variety of silicas and organosilicas with cylindrical mesopores