Smc5/6 is required for repair at collapsed replication forks.

In eukaryotes, three pairs of structural-maintenance-of-chromosome (SMC) proteins are found in conserved multisubunit protein complexes required for chromosomal organization. Cohesin, the Smc1/3 complex, mediates sister chromatid cohesion while two condensin complexes containing Smc2/4 facilitate chromosome condensation. Smc5/6 scaffolds an essential complex required for homologous recombination repair. We have examined the response of smc6 mutants to the inhibition of DNA replication. We define homologous recombination-dependent and -independent functions for Smc6 during replication inhibition and provide evidence for a Rad60-independent function within S phase, in addition to a Rad60-dependent function following S phase. Both genetic and physical data show that when forks collapse (i.e., are not stabilized by the Cds1Chk2 checkpoint), Smc6 is required for the effective repair of resulting lesions but not for the recruitment of recombination proteins. We further demonstrate that when the Rad60-dependent, post-S-phase Smc6 function is compromised, the resulting recombination-dependent DNA intermediates that accumulate following release from replication arrest are not recognized by the G2/M checkpoint

Smc5/6 maintains stalled replication forks in a recombination-competent conformation

The Smc5/6 structural maintenance of chromosomes complex is required for efficient homologous recombination (HR). Defects in Smc5/6 result in chromosome missegregation and fragmentation. By characterising two Schizosaccharomyces pombe smc6 mutants, we define two separate functions for Smc5/6 in HR. The first represents the previously described defect in processing recombination-dependent DNA intermediates when replication forks collapse, which leads to increased rDNA recombination. The second novel function defines Smc5/6 as a positive regulator of recombination in the rDNA and correlates mechanistically with a requirement to load RPA and Rad52 onto chromatin genome-wide when replication forks are stably stalled by nucleotide depletion. Rad52 is required for all HR repair, but Rad52 loading in response to replication fork stalling is unexpected and does not correlate with damage-induced foci. We propose that Smc5/6 is required to maintain stalled forks in a stable recombination-competent conformation primed for replication restart

Visual localization in challenging environments

Visual localization, the method of self-localization based on camera images, has established as an additional, GNSS-free technology that is investigated in increasingly real and challenging applications. Particularly demanding is the self-localization of first responders in unstructured and unknown environments, for which visual localization can substantially contribute to increase the situational awareness and safety of first responders. Challenges arise from the operation under adverse conditions on computationally restricted platforms in the presence of dynamic objects. Current solutions are quickly pushed to their limits and the development of more robust approaches is of high demand. This thesis investigates the application of visual localization in dynamic, adverse environments to identify challenges and accordingly to increase the robustness, on the example of a dedicated visual-inertial navigation system. The methodical contributions of this work relate to the introduction of semantic understanding, improvements in error propagation and the development of a digital twin. The geometric visual odometry component is extended to a hybrid approach that includes a deep neural network for semantic segmentation to ignore distracting image areas of certain object classes. A Sensor-AI approach complements this method by directly training the network to segment image areas that are critical for the considered visual odometry system. Another improvement results from analyses and modifications of the existing error propagation in visual odometry. Furthermore, a digital twin is presented that closely replicates geometric and radiometric properties of the real sensor system in simulation in order to multiply experimental possibilities. The experiments are based on datasets from inspections that are used to motivate three first responder scenarios, namely indoor rescue, flood disaster and wildfire. The datasets were recorded in corridor, mall, coast, river and fumarole environments and aim to analyze the influence of the dynamic elements person, water and smoke. Each investigation starts with extensive in-depth analyses in simulation based on created synthetic video clones of the respective dynamic environments. Specifically, a combined sensitivity analysis allows to jointly consider environment, system design, sensor property and calibration error parameters to account for adverse conditions. All investigations are verified with experiments based on the real system. The results show the susceptibility of geometric approaches to dynamic objects in challenging scenarios. The introduction of the segmentation aid within the hybrid system contributes well in terms of robustness by preventing significant failures, but understandably it cannot compensate for a lack of visible static backgrounds. As a consequence, future visual localization systems require both the ability of semantic understanding and its integration into a complementary multi-sensor system

Orthogonal enzyme-driven timers for DNA strand displacement reactions

Here, we demonstrate a strategy to rationally program a delayed onset of toehold-mediated DNA strand displacement reactions (SDRs). The approach is based on blocker strands that efficiently inhibit the strand displacement by binding to the toehold domain of the target DNA. Specific enzymatic degradation of the blocker strand subsequently enables SDR. The kinetics of the blocker enzymatic degradation thus controls the time at which the SDR starts. By varying the concentration of the blocker strand and the concentration of the enzyme, we show that we can finely tune and modulate the delayed onset of SDR. Additionally, we show that the strategy is versatile and can be orthogonally controlled by different enzymes each specifically targeting a different blocker strand. We designed and established three different delayed SDRs using RNase H and two DNA repair enzymes (formamidopyrimidine DNA glycosylase and uracil-DNA glycosylase) and corresponding blockers. The achieved temporal delay can be programed with high flexibility without undesired leak and can be conveniently predicted using kinetic modeling. Finally, we show three possible applications of the delayed SDRs to temporally control the ligand release from a DNA nanodevice, the inhibition of a target protein by a DNA aptamer, and the output signal generated by a DNA logic circuit

One amino acid makes the difference: the formation of ent-kaurene and 16alpha-hydroxy-ent-kaurane by diterpene synthases in poplar

BACKGROUND: Labdane-related diterpenoids form the largest group among the diterpenes. They fulfill important functions in primary metabolism as essential plant growth hormones and are known to function in secondary metabolism as, for example, phytoalexins. The biosynthesis of labdane-related diterpenes is mediated by the action of class II and class I diterpene synthases. Although terpene synthases have been well investigated in poplar, little is known about diterpene formation in this woody perennial plant species. RESULTS: The recently sequenced genome of Populus trichocarpa possesses two putative copalyl diphosphate synthase genes (CPS, class II) and two putative kaurene synthase genes (KS, class I), which most likely arose through a genome duplication and a recent tandem gene duplication, respectively. We showed that the CPS-like gene PtTPS17 encodes an ent-copalyl diphosphate synthase (ent-CPS), while the protein encoded by the putative CPS gene PtTPS18 showed no enzymatic activity. The putative kaurene synthases PtTPS19 and PtTPS20 both accepted ent-copalyl diphosphate (ent-CPP) as substrate. However, despite their high sequence similarity, they produced different diterpene products. While PtTPS19 formed exclusively ent-kaurene, PtTPS20 generated mainly the diterpene alcohol, 16α-hydroxy-ent-kaurane. Using homology-based structure modeling and site-directed mutagenesis, we demonstrated that one amino acid residue determines the different product specificity of PtTPS19 and PtTPS20. A reciprocal exchange of methionine 607 and threonine 607 in the active sites of PtTPS19 and PtTPS20, respectively, led to a complete interconversion of the enzyme product profiles. Gene expression analysis revealed that the diterpene synthase genes characterized showed organ-specific expression with the highest abundance of PtTPS17 and PtTPS20 transcripts in poplar roots. CONCLUSIONS: The poplar diterpene synthases PtTPS17, PtTPS19, and PtTPS20 contribute to the production of ent-kaurene and 16α-hydroxy-ent-kaurane in poplar. While ent-kaurene most likely serves as the universal precursor for gibberellins, the function of 16α-hydroxy-ent-kaurane in poplar is not known yet. However, the high expression levels of PtTPS20 and PtTPS17 in poplar roots may indicate an important function of 16α-hydroxy-ent-kaurane in secondary metabolism in this plant organ. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-015-0647-6) contains supplementary material, which is available to authorized users

Cultural Heritage, Sustainable Development, and Climate Policy: Comparing the UNESCO World Heritage Cities of Potsdam and Bern

Developing sustainable, carbon-neutral, and climate-resilient districts seems to be particularly challenging with respect to historic city centers. However, barriers posed by legal requirements for historical buildings are counterbalanced by opportunities because historic cities have not undergone urban modernization and did not embrace the concept of functional cities, which nowadays impedes urban sustainability transformations. Thus, this paper focuses on the relationship between cultural heritage, urban sustainable development, and climate policy. We study continuity and change in the mid-sized UNESCO World Heritage cities Potsdam (Germany) and Bern (Switzerland). These matching forerunner cities share many characteristics, which enables them to transfer policies and jointly create new solutions for common problems. We find that national context matters, but we also identify functional equivalents like referenda and active citizen participation. Despite many similarities, Potsdam is ahead of Bern with respect to the institutionalization and integration of climate mitigation and adaptation. The comparative analysis (interviews and document analysis) identifies innovations that can be transferred between the two cities (e.g., Potsdam’s integrative climate policy or Bern’s efforts to become a role model for stakeholders and citizens). Moreover, the challenge to coordinate heritage management and climate governance offers chances for cooperation between matching cities like Bern and Potsda

The organ-specific expression of terpene synthase genes contributes to the terpene hydrocarbon composition of chamomile essential oils

BACKGROUND: The essential oil of chamomile, one of the oldest and agronomically most important medicinal plant species in Europe, has significant antiphlogistic, spasmolytic and antimicrobial activities. It is rich in chamazulene, a pharmaceutically active compound spontaneously formed during steam distillation from the sesquiterpene lactone matricine. Chamomile oil also contains sesquiterpene alcohols and hydrocarbons which are produced by the action of terpene synthases (TPS), the key enzymes in constructing terpene carbon skeletons. RESULTS: Here, we present the identification and characterization of five TPS enzymes contributing to terpene biosynthesis in chamomile (Matricaria recutita). Four of these enzymes were exclusively expressed in above-ground organs and produced the common terpene hydrocarbons (−)-(E)-β-caryophyllene (MrTPS1), (+)-germacrene A (MrTPS3), (E)-β-ocimene (MrTPS4) and (−)-germacrene D (MrTPS5). A fifth TPS, the multiproduct enzyme MrTPS2, was mainly expressed in roots and formed several Asteraceae-specific tricyclic sesquiterpenes with (−)-α-isocomene being the major product. The TPS transcript accumulation patterns in different organs of chamomile were consistent with the abundance of the corresponding TPS products isolated from these organs suggesting that the spatial regulation of TPS gene expression qualitatively contribute to terpene composition. CONCLUSIONS: The terpene synthases characterized in this study are involved in the organ-specific formation of essential oils in chamomile. While the products of MrTPS1, MrTPS2, MrTPS4 and MrTPS5 accumulate in the oils without further chemical alterations, (+)-germacrene A produced by MrTPS3 accumulates only in trace amounts, indicating that it is converted into another compound like matricine. Thus, MrTPS3, but also the other TPS genes, are good markers for further breeding of chamomile cultivars rich in pharmaceutically active essential oils