Carbon and Nitrogen Mineralization of Lead Treated Soils in the Eastern Mediterranean Region,Turkey

The aim of this study was to determine the first effect of lead on microbial activity in soil. The study was carried out in the soil samples from four different radish (Raphanus sativus L. var. radicula, Brassicaceae) fields along the highway in a district (Kadirli, Osmaniye) of the Eastern Mediterranean Region, Turkey. After the calculation of Pb contents, the Pb amounts of the soil samples were brought up to 50 and 100 mg Pb kg^?1 by treatment with Pb(NO ₃ ) ₂ , and the samples for the carbon and the nitrogen mineralization were incubated under controlled conditions (28°C, constant moist). The carbon mineralization was determined by a CO ₂ respiration method for 30 days. The nitrogen mineralization was observed in vitro for 6 weeks. The untreated group was statistically different from the 50 and 100 mg Pb kg^?1 treatments in the aspect of the C(CO ₂ ) outlet during mineralization (P ≤ 0.05), but difference between the 50 and 100 mg Pb kg^?1 treatments was not significant. NH ₄ -N and NO ₃ -N contents of each soil were shown differences between across treatments. Based on these results, it is possible to conclude that the addition of 50 and 100 mg Pb kg^?1 provided a toxic effect threshold for the microbial activity into 30 days.     

Incidence of mycoflora and mycotoxins in some edible fruits and seeds of forest origin

Twelve fungi namelyAlternaria alternata, Aspergillus flavus, A niger, A ochraceus, Actinomucor repens, Capnodoium spp., Curvularia lunata, Fusarium pallidoroseum, F solani, F verticillioides, Penicillium citrinum and Rhizopus stolonifer were recorded from samples ofAegle marmelos, Aesculus indica, Buchanania lanzan andPinus gerardiana. In case ofPrunus amygdalus only Rstolonifer was recorded. A significant variation in pattern of mycoflora incidence was observed in terms of source and season. Fungal infestation in most of the substrates was found to be highest during monsoon. Aflatoxins were the most common mycotoxins elaborated by different isolates ofA flavus obtained fromA marmelos, B lanzan andP gerardiana. The amount of aflatoxins produced by the toxigenic isolates ofA flavus was in the range of traces to 0.9–26.0 μg/ml inA marmelos, 0.8–17.5 μg/ml inP gerardiana and 0.65–13.2 μg/ml inB lanzan. The percentage toxigenicity was comparatively lower in the isolates of other mycotoxigenic fungi. Aflatoxins were detected almost in all the samples analyzed for mycotoxin contamination. However, traces of zearalenone were detected inA marmelos. The concentration of aflatoxin B₁ was in the range of 0.13–0.75 μg/g inA marmelos, 0.09–0.60 μg/g inP gerardiana and 0.01–0.20 ug/g inB lanzan. Mycotoxins were not detected inAesculus indica andPrunus amygdalus.     

BRCA2 diffuses as oligomeric clusters with RAD51 and changes mobility after DNA damage in live cells

Genome maintenance by homologous recombination depends on coordinating many proteins in time and space to assemble at DNA break sites. To understand this process, we followed the mobility of BRCA2, a critical recombination mediator, in live cells at the single-molecule level using both single-particle tracking and fluorescence correlation spectroscopy. BRCA2-GFP and -YFP were compared to distinguish diffusion from fluorophore behavior. Diffusive behavior of fluorescent RAD51 and RAD54 was determined for comparison. All fluorescent proteins were expressed from endogenous loci. We found that nuclear BRCA2 existed in oligomeric clusters, and exhibited heterogeneous mobility. DNA damage increased BRCA2 transient binding, presumably including binding to damaged sites. Despite its very different size, RAD51 displayed mobility similar to BRCA2, which indicates physical interaction between these proteins both before and after induction of DNA damage. We propose that BRCA2-mediated sequestration of nuclear RAD51 serves to prevent inappropriate DNA interactions and that all RAD51 is delivered to DNA damage sites in association with BRCA2.     

Mediators of Homologous DNA Pairing

Homologous DNA pairing and strand exchange are at the core of homologous recombination. These reactions are promoted by a DNA-strand-exchange protein assembled into a nucleoprotein filament comprising the DNA-pairing protein, ATP, and single-stranded DNA. The catalytic activity of this molecular machine depends on control of its dynamic instability by accessory factors. Here we discuss proteins known as recombination mediators that facilitate formation and functional activation of the DNA-strand-exchange protein filament. Although the basics of homologous pairing and DNA-strand exchange are highly conserved in evolution, differences in mediator function are required to cope with differences in how single-stranded DNA is packaged by the single-stranded DNA-binding protein in different species, and the biochemical details of how the different DNA-strand-exchange proteins nucleate and extend into a nucleoprotein filament. The set of (potential) mediator proteins has apparently expanded greatly in evolution, raising interesting questions about the need for additional control and coordination of homologous recombination in more complex organisms.Homologous recombination, the exchange of base pairing between homologous DNA molecules, is a central process for life (Morrical 2014). Not only does it create genetic diversity that sustains a population, it is essential at the cellular level for proper replication and maintenance of genomes (Wyman and Kanaar 2006). Given this central role in DNA metabolism, it is not surprising that the core reaction of homologous recombination is highly conserved among all kingdoms of life. The fundamental unit of this reaction is a DNA-strand-exchange protein, a small ATP-binding protein of ∼40 kDa. The DNA-strand-exchange protomers assemble head-to-tail in a right-handed helical filament around single-stranded (ss) DNA. This molecular assembly, programmed by the sequence of the bases of the bound DNA strand, recognizes homology in a double-stranded (ds) partner DNA molecule. Through protein-mediated manipulation of DNA structure and disassembly of protein–protein and protein–DNA interactions, exchange of base-pairing partners is achieved (Wyman 2011; Jasin and Rothstein 2013).It is essential that the DNA-strand-exchange reaction at the core of homologous recombination is regulated such that it is actively applied in specific and changing conditions. Inappropriate DNA rearrangements also need to be avoided where they would be disastrous rather than beneficial. Thus, there need to be tipping points at which the reaction can either be driven forward or reversed, an important feature required to attain quality control (Kanaar et al. 2008). To achieve intricate levels of regulation, reaction choreographers have evolved, which are often referred to as positive or negative recombination mediators or effectors (Daley et al. 2014). Interestingly, although the fundamentals of the DNA-strand-exchange reaction are the same for bacteriophages, bacteria, archaea, and eukaryotes (Maher et al. 2011; White 2011), it appears that the set of proteins that influences homologous recombination has expanded significantly during evolution of more complex life forms (Fig. 1). In this review, we focus on positive recombination mediators of the highly conserved DNA-strand-exchange protein proteins UvsX (bacteriophages), RecA (bacteria), and RAD51 (eukaryotes).Open in a separate windowFigure 1.The figure shows the increasing evolutionary complexity within the group of homologous recombination accessory factors that contribute to the formation and stability of the DNA-strand-exchange protein nucleoprotein filament in the key model organisms. Main homologous recombination steps (resection [Symington 2014], coating with ssDNA-binding protein [S], loading of the DNA-strand-exchange protein [R], and strand invasion) are shown schematically. Phylogenetic relationships between homologous proteins are indicated with solid lines in cases of well-supported orthology or broken lines when the exact evolutionary relationship is uncertain; x indicates no close homolog in a fully sequenced genome. Because phage, bacterial, and archaeal homologous recombination accessory proteins do not show detectably sequence similarity and have likely evolved independently, they are displayed as separate domains. Proteins that most closely meet the “mediator” definition criteria are indicated in bold. ∼ indicates the ability to promote annealing of protein-coated ssDNA, shared by UvsY, RecO, and RAD52, which is a remarkable example of convergent evolution emphasizing the universal usefulness of this biochemical activity.At the core of homologous recombination is a DNA-strand-exchange protein–ATP–ssDNA nucleoprotein filament (Wyman 2011). The positive recombination mediator proteins facilitate formation and functional activation of this molecular machinery. Specifically, in the original definition, recombination mediator proteins are described to facilitate loading DNA-strand-exchange proteins onto ssDNA that is coated by ssDNA-binding proteins (Beernink and Morrical 1999). Mediators influence the competition for ssDNA binding in favor of the filament forming DNA-strand-exchange protein. Mediators also influence the preferential binding of DNA-strand-exchange proteins to ssDNA in favor of the much more abundant dsDNA in cells. Although in biochemical assays the order of addition of protein can be controlled by the experimenter, with variable results for efficiency of assay outcome, in vivo exposed ssDNA will be rapidly and effectively bound by abundant, high-affinity ssDNA-binding proteins (Dickey et al. 2013). These proteins need to be prevented from binding or replaced by the DNA-strand-exchange protein to initiate homologous recombination. However, in part because of the semantic ambiguity of the term and the large number of factors affecting homologous recombination that still lack a clearly established biochemical function, the term mediator is often applied more generally in the literature. We prefer the term “homologous recombination accessory proteins” for this broader class of proteins, and will focus our discussion here on those mediators that fit the original definition when possible. A model for mediator-assisted sequential handover of ssDNA from the ssDNA-binding protein to the DNA-strand-exchange protein gradually emerged from studies of the slimmed down recombination systems, especially phage T4. The properties attributed to mediators include: ssDNA binding, interaction with the ssDNA-binding protein, interaction with the DNA-strand-exchange protein, and “filament stabilization” by affecting ATPase activity of the nucleoprotein filament (Liu et al. 2011a). This last feature, an effect on nucleotide exchange or ATPase activity by the nucleoprotein filament, became the template used to search for candidate mediators in higher organisms. Typically, the potential mediator proteins were then tested for the specific interactions and functions needed to promote DNA-strand-exchange protein filament formation and activation.     

Caffeine suppresses homologous recombination through interference with RAD51-mediated joint molecule formation

Caffeine is a widely used inhibitor of the protein kinases that play a central role in the DNA damage response. We used chemical inhibitors and genetically deficient mouse embryonic stem cell lines to study the role of DNA damage response in stable integration of the transfected DNA and found that caffeine rapidly, efficiently and reversibly inhibited homologous integration of the transfected DNA as measured by several homologous recombination-mediated gene-targeting assays. Biochemical and structural biology experiments revealed that caffeine interfered with a pivotal step in homologous recombination, homologous joint molecule formation, through increasing interactions of the RAD51 nucleoprotein filament with non-homologous DNA. Our results suggest that recombination pathways dependent on extensive homology search are caffeine-sensitive and stress the importance of considering direct checkpoint-independent mechanisms in the interpretation of the effects of caffeine on DNA repair.