We present the Flp-nick system, which allows introduction of a protein-bound nick at a single genomic site in Saccharomyces cerevisiae and thus mimics a stabilized topoisomerase I-DNA cleavage complex. We took advantage of a mutant Flp recombinase that can introduce a nick at a specific Flp recombinase recognition target site that has been integrated in the yeast genome. The genetic requirement for cells to cope with this insult is the same as for cells treated with camptothecin, which traps topoisomerase I-DNA cleavage complexes genome-wide. Hence, a single protein-bound nick is enough to kill cells if functional repair pathways are lacking. The Flp-nick system can be used to dissect repair, checkpoint and replication fork management pathways activated by a single genomic insult, and it allows the study of events at the damage site, which so far has been impossible to address.

Characterization of local order in thin films is challenging with pair distribution function (PDF) analysis because of the minute mass of the scattering material. Here, it is demonstrated that reliable high-energy grazing-incidence total X-ray scattering data can be obtained in situ during thin-film deposition by radio-frequency magnetron sputtering. A benchmark system of Pt was investigated in a novel sputtering chamber mounted on beamline P07-EH2 at the PETRA III synchrotron. Robust and high-quality PDFs can be obtained from films as thin as 3 nm and atomistic modelling of the PDFs with a time resolution of 0.5 s is possible. In this way, it was found that a polycrystalline Pt thin film deposits with random orientation at 8 W and 2 × 10−2 mbar at room temperature. From the PDF it was found that the coherent-scattering domains grow with time. While the first layers are formed with a small tensile strain this relaxes towards the bulk value with increasing film thickness.

To address how eukaryotic replication forks respond to fork stalling caused by strong non-covalent protein–DNA barriers, we engineered the controllable Fob-block system in Saccharomyces cerevisiae. This system allows us to strongly induce and control replication fork barriers (RFB) at their natural location within the rDNA. We discover a pivotal role for the MRX (Mre11, Rad50, Xrs2) complex for fork integrity at RFBs, which differs from its acknowledged function in double-strand break processing. Consequently, in the absence of the MRX complex, single-stranded DNA (ssDNA) accumulates at the rDNA. Based on this, we propose a model where the MRX complex specifically protects stalled forks at protein–DNA barriers, and its absence leads to processing resulting in ssDNA. To our surprise, this ssDNA does not trigger a checkpoint response. Intriguingly, however, placing RFBs ectopically on chromosome VI provokes a strong Rad53 checkpoint activation in the absence of Mre11. We demonstrate that proper checkpoint signalling within the rDNA is restored on deletion of SIR2. This suggests the surprising and novel concept that chromatin is an important player in checkpoint signalling.

Faithful DNA replication with correct termination is essential for genome stability and transmission of genetic information. Here we have investigated the potential roles of Topoisomerase II (Top2) and the RecQ helicase Sgs1 during late stages of replication. We find that cells lacking Top2 and Sgs1 (or Top3) display two different characteristics during late S/G2 phase, checkpoint activation and accumulation of asymmetric X-structures, which are both independent of homologous recombination. Our data demonstrate that checkpoint activation is caused by a DNA structure formed at the strongest rDNA replication fork barrier (RFB) during replication termination, and consistently, checkpoint activation is dependent on the RFB binding protein, Fob1. In contrast, asymmetric X-structures are formed independent of Fob1 at less strong rDNA replication fork barriers. However, both checkpoint activation and formation of asymmetric X-structures are sensitive to conditions, which facilitate fork merging and progression of replication forks through replication fork barriers. Our data are consistent with a redundant role of Top2 and Sgs1 together with Top3 (Sgs1-Top3) in replication fork merging at rDNA barriers. At RFB either Top2 or Sgs1-Top3 is essential to prevent formation of a checkpoint activating DNA structure during termination, but at less strong rDNA barriers absence of the enzymes merely delays replication fork merging, causing an accumulation of asymmetric termination structures, which are solved over time.

Precursor structures (PSs) in solution are expected to influenceb oth nanocrystal formation mechanisms, as well as crystallization of specific polymorphs. Herein, Group 13 PS structures determined by paird istribution functiona nd extended X-ray absorption fine structure analysis are reported.C orner-sharing octahedral dimers form from the metal nitrates dissolved in either water,i sopropanol, or ethanol at room temperature contradicting previous studies that suggested monomers or larger Keggin clusters. Because all crystalline indium oxides have octahedral coordination, crystalsc an easily nucleate from the observed PSs. Similarly,M OOH (M = Al and Ga) with octahedral Mc oordination is expected to form readily from the PSs, whereas formation of g-M 2 O 3 requires ap artial conversion to tetrahedral Mcoordination. This explains the long-standing observation of initial AlOOHf ormation as ab ottleneck for g-Al 2 O 3 synthesis. Different indium polymorphs crystallize from the variouss olvents, and thus there is no obvious link between the PSs and observed polymorphism.The Group 13 oxides are extremely important in technological applications. [1] Indeed, huge amounts of g-Al 2 O 3 are produced every month worldwide as catalyst support material, watertreatment agent, or image-enhancementa dditive, [1a, 2] whereas Ga 2 O 3 and In 2 O 3 are essential materials, for example, in the semiconductor industry. [1b,c] Al, Ga, and In form relatedo xides and hydroxides, such as M(OH) 3 ,MOOH and M 2 O 3 ,but the individual systems exhibit complex polymorphism with many different crystal structures. In general, the physicala nd chemical properties are controlled not only by the exact crystals tructure, but also the crystal morphology and size, [1a] and it is challenging to achieve control of all these characteristics. For all three systems, it is possible to calcine MOOH to form various M 2 O 3 polymorphs, but for industrial applications,i ti sd esirable to avoid the energy-consuming calcination step.M oreover,t he resultingm orphologya nd size of the M 2 O 3 crystals typically reflect the intermediate MOOH, and in addition high-temperatures fosters crystalliteg rowth, which further limits the control. Thus,d irect synthesis pathways are targeted. This wasa chieved by Lock et al. [3] andN oguchi et al. [4] who reported onestep synthesis of g-Al 2 O 3 (withint he time resolution of the experiments) at very specific solvent conditions or at very high reactiont emperature.C orrelation between the water content/ solventa nd the resulting oxide phase has also been reported for the In system. [5] However, to controla nd further develop Group13 oxide synthesis, atomics cale understandingo ft he crystal formation processes is required.During the last decade it has becomei ncreasingly clear that classical crystal nucleation theory established for supersaturated solutions [6] and homogenous melts [7] is too simplistic in relation to the complex chemical solutione nvironments presenti n formation of many modernm aterials. [8] Understan...

The conserved family of RecQ DNA helicases consists of caretaker tumour suppressors, that defend genome integrity by acting on several pathways of DNA repair that maintain genome stability. In budding yeast, Sgs1 is the sole RecQ helicase and it has been implicated in checkpoint responses, replisome stability and dissolution of double Holliday junctions during homologous recombination. In this study we investigate a possible genetic interaction between SGS1 and RAD9 in the cellular response to methyl methane sulphonate (MMS) induced damage and compare this with the genetic interaction between SGS1 and RAD24. The Rad9 protein, an adaptor for effector kinase activation, plays well-characterized roles in the DNA damage checkpoint response, whereas Rad24 is characterized as a sensor protein also in the DNA damage checkpoint response. Here we unveil novel insights into the cellular response to MMS-induced damage. Specifically, we show a strong synergistic functionality between SGS1 and RAD9 for recovery from MMS induced damage and for suppression of gross chromosomal rearrangements, which is not the case for SGS1 and RAD24. Intriguingly, it is a Rad53 independent function of Rad9, which becomes crucial for genome maintenance in the absence of Sgs1. Despite this, our dissection of the MMS checkpoint response reveals parallel, but unequal pathways for Rad53 activation and highlights significant differences between MMS- and hydroxyurea (HU)-induced checkpoint responses with relation to the requirement of the Sgs1 interacting partner Topoisomerase III (Top3). Thus, whereas earlier studies have documented a Top3-independent role of Sgs1 for an HU-induced checkpoint response, we show here that upon MMS treatment, Sgs1 and Top3 together define a minor but parallel pathway to that of Rad9.

Prussian White (PW, Na2Fe[Fe(CN)6]∙zH2O) is a promising cathode material for use in sodium-ion batteries (SIBs) for large-scale energy storage applications which demand long cycling life-times. For use in non-aqueous battery applications PW must not contain any water, and yet its removal induces a large volume change destabilizing the structure and reducing cycling life. Indeed, the material undergoes multiple phase transitions depending upon both the sodium and water content. Due to the extensive use of X ray diffraction, the mechanism behind the observed phase transitions is poorly understood. Here, we show a neutron diffraction study exploring the influence of water on the structure of PW. For the first time, two temperature dependent and composition independent structures were observed near room temperature which differ in the FeN6 and FeC6 octahedral tilting configurations. The fundamental nature of this distortion, which drives the large volume changes, was determined to be connected to water ordering in the framework. Further, using distortion mode analysis during the dehydration process, it was determined that water does not modify the nature of the distortions present. Rather, water was found to modulate the magnitude of pre-existing structural distortions. These results provide a robust fundamental understanding to the chemical driving forces impacting the nature and magnitude of structural distortions in PBAs. The insight provides guidance for designing and quantifying tilt-engineering in PBAs ultimately enabling new materials with enhanced long-term electrochemical performance in battery applications.

The indium oxides, c-In2O3, h-In2O3, InOOH and In(OH)3, constitute an important class of wide band gap semiconductors. Synthesis of any indium oxide phase involves manoeuvring in a complex matrix of...