Topoisomerase 3α and RMI1 Suppress Somatic Crossovers and Are Essential for Resolution of Meiotic Recombination Intermediates in Arabidopsis thaliana

Topoisomerases are enzymes with crucial functions in DNA metabolism. They are ubiquitously present in prokaryotes and eukaryotes and modify the steady-state level of DNA supercoiling. Biochemical analyses indicate that Topoisomerase 3α (TOP3α) functions together with a RecQ DNA helicase and a third partner, RMI1/BLAP75, in the resolution step of homologous recombination in a process called Holliday Junction dissolution in eukaryotes. Apart from that, little is known about the role of TOP3α in higher eukaryotes, as knockout mutants show early lethality or strong developmental defects. Using a hypomorphic insertion mutant of Arabidopsis thaliana (top3α-2), which is viable but completely sterile, we were able to define three different functions of the protein in mitosis and meiosis. The top3α-2 line exhibits fragmented chromosomes during mitosis and sensitivity to camptothecin, suggesting an important role in chromosome segregation partly overlapping with that of type IB topoisomerases. Furthermore, AtTOP3α, together with AtRECQ4A and AtRMI1, is involved in the suppression of crossover recombination in somatic cells as well as DNA repair in both mammals and A. thaliana. Surprisingly, AtTOP3α is also essential for meiosis. The phenotype of chromosome fragmentation, bridges, and telophase I arrest can be suppressed by AtSPO11 and AtRAD51 mutations, indicating that the protein is required for the resolution of recombination intermediates. As Atrmi1 mutants have a similar meiotic phenotype to Attop3α mutants, both proteins seem to be involved in a mechanism safeguarding the entangling of homologous chromosomes during meiosis. The requirement of AtTOP3α and AtRMI1 in a late step of meiotic recombination strongly hints at the possibility that the dissolution of double Holliday Junctions via a hemicatenane intermediate is indeed an indispensable step of meiotic recombination.     

Interplay of RNA Elements in the Dengue Virus 5′ and 3′ Ends Required for Viral RNA Replication

Dengue virus (DENV) is a member of the Flavivirus genus of positive-sense RNA viruses. DENV RNA replication requires cyclization of the viral genome mediated by two pairs of complementary sequences in the 5′ and 3′ ends, designated 5′ and 3′ cyclization sequences (5′-3′ CS) and the 5′ and 3′ upstream of AUG region (5′-3′ UAR). Here, we demonstrate that another stretch of six nucleotides in the 5′ end is involved in DENV replication and possibly genome cyclization. This new sequence is located downstream of the AUG, designated the 5′ downstream AUG region (5′ DAR); the motif predicted to be complementary in the 3′ end is termed the 3′ DAR. In addition to the UAR, CS and DAR motifs, two other RNA elements are located at the 5′ end of the viral RNA: the 5′ stem-loop A (5′ SLA) interacts with the viral RNA-dependent RNA polymerase and promotes RNA synthesis, and a stem-loop in the coding region named cHP is involved in translation start site selection as well as RNA replication. We analyzed the interplay of these 5′ RNA elements in relation to RNA replication, and our data indicate that two separate functional units are formed; one consists of the SLA, and the other includes the UAR, DAR, cHP, and CS elements. The SLA must be located at the 5′ end of the genome, whereas the position of the second unit is more flexible. We also show that the UAR, DAR, cHP, and CS must act in concert and therefore likely function together to form the tertiary RNA structure of the circularized DENV genome.Dengue virus (DENV), a member of the Flaviviridae family, is a human pathogen causing dengue fever, the most common mosquito-borne viral disease in humans. The virus has become a major international public health concern, with 3 billion people at risk for infection and an estimated 50 million dengue cases worldwide every year (28). Neither specific antiviral therapies nor licensed vaccines are available, and the biology of the virus is poorly understood.DENV is a small enveloped virus containing a positive-stranded RNA genome with a length of approximately 10.7 kb. The virus encodes one large polyprotein that is co- and posttranslationally cleaved into 10 viral proteins. The structural proteins C, prM/M, and E are located in the N terminus, followed by the nonstructural proteins NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5 (6, 10). NS5, the largest of the viral proteins, functions as an RNA-dependent RNA polymerase (RdRP) (29). The coding region is flanked at both ends by untranslated regions (UTR). The 5′ end has a type I cap structure (m⁷GpppAmp) mediating cap-dependent translation, but the virus can switch to a noncanonical translation mechanism under conditions in which translation factors are limiting (13). Cellular mRNAs are known to circularize via a protein-protein bridge between eIF4G and eIF4E (the cap binding complex) at the 5′ end and the poly(A) binding protein (PABP) at the 3′ end, enhancing translation efficiency. Despite the fact that the DENV 3′ UTR lacks a poly(A) tail, recent findings demonstrated binding of PABP to the 3′ UTR and an effect on RNA translation, suggesting a similar mechanism (12, 26).In addition to a presumed protein-mediated genome circularization regulating viral translation, an RNA-RNA-based 5′ and 3′ (5′-3′) end interaction, which can occur in the absence of proteins, leads to circularization of the viral genome (1, 3, 4, 18, 20, 30, 33, 34). This cyclization of the genome is necessary for viral RNA replication, and thus far, two complementary sequences at the 5′ and 3′ ends have been identified (3). The first are the cyclization sequences (CS) present in the capsid-coding region at the 5′ end (5′ CS) and upstream of the 3′ stem-loop (3′ SL) in the 3′ UTR (3′ CS) (2, 4, 18, 20, 30). A second sequence, known as the 5′ upstream AUG region (5′ UAR) element in the 5′ UTR, base pairs with its complementary 3′ UAR counterpart, which is located at the bottom part of 3′ SL (1, 4, 30). Recently, the structure of the 5′ end of the DENV genome hybridized to the 3′ end was determined in solution (25), confirming previous computer-predicted structures for genome cyclization (4, 20, 30). Besides the base pairing between 5′-3′ UAR and 5′-3′ CS sequences, a third stretch of nucleotides was identified to form a double-stranded (ds) region between the 5′ and 3′ ends.In addition to RNA sequences involved in 5′-3′-end interactions that are necessary for cyclization, the 5′ end of the viral genome harbors at least two more functional RNA elements, the stem-loop A (SLA) and capsid-coding region hairpin (cHP). The SLA consists of the first 70 nucleotides (nt) of the genome, forming a stable stem-loop structure. This structure has been confirmed in several studies and identified as a promoter element for RNA synthesis that recruits the viral RdRp NS5 (16, 22). Once NS5 is bound to the SLA at the 5′ end, it is believed to be delivered to the initiation site of minus-strand RNA synthesis at the 3′ end via 5′-3′ RNA-RNA circularization. In addition, a short poly(U) tract located immediately downstream of SLA has been shown to be necessary for RNA synthesis, although it is not involved in genome circularization (22). Finally, the cHP element resides within the capsid-coding region; it directs start codon selection and is essential for RNA replication (8, 9). The cHP structure is more important than its primary sequence. For start codon selection, it is believed that the cHP stalls the scanning initiation complex over the first AUG, favoring its recognition (9). In the case of RNA replication, the cHP likely stabilizes the overall 5′-3′ panhandle structure or participates in recruitment of factors associated with the replicase machinery (8).In this study, we demonstrate that in addition to the 5′ CS and 5′ UAR sequences, a third stretch of nucleotides in the 5′ end is required for RNA replication and appears to be involved in genome circularization. This new motif is located downstream of the AUG and was therefore designated the downstream AUG region (5′ DAR) element, with the predicted counterpart in the 3′ end designated the 3′ DAR. Our results indicate that the 5′ DAR modulates RNA-RNA interaction and RNA replication, and restoring complementarity between the 5′ DAR and 3′ DAR rescues detrimental effects caused by mutations in the 5′ DAR on genome circularization and RNA replication. Although the role of the predicted 3′ DAR counterpart is less conclusive, it may serve to make other structures and sequences in the 3′ end available for 5′-3′ RNA-RNA interaction to facilitate the replication-competent conformation of the DENV genome.Furthermore, we analyzed the functional interplay of RNA elements in the viral 5′ end, showing that two separate units are formed during replication. The first consists of the SLA, and it must be located at the very 5′ end of the genome. The second unit includes UAR, DAR, cHP, and CS elements, and the positional requirements are more flexible within the DENV RNA 5′ terminus. However, all four elements in the second unit must act in concert, forming a functional tertiary RNA structure of the circularized viral genome.     

Zinc Deficiency Enhances the Induction of Micronuclei and 8-Hydroxy-2′-Deoxyguanosine Via Superoxide Radical in Bone Marrow of Zinc-Deficient Rats

The aim of the present study was to examine whether zinc (Zn) deficiency augmented the frequency of micronuclei, an indicator of chromosome aberration, and the induction of 8-hydroxy-2′-deoxyguanosine (8-OHdG), a marker of cellular DNA damage derived from oxidative stress, in rat bone marrow cells or not. Both the frequency of micronuclei and the induction of 8-OHdG were significantly increased in rats fed with a Zn-deficient versus a standard diet for 6 weeks (p?<?0.005). The supplementation of Zn with a standard diet for 4 weeks to rats fed with a Zn-deficient diet for 6 weeks restored the enhanced induction of micronuclei and 8-OHdG to levels comparable to those seen in rats fed with a standard diet for 10 weeks, indicating that the shortage of Zn in the body is involved in the induction of micronuclei and 8-OHdG. Again, the membrane-permeable superoxide dismutase mimetic superoxide scavenger, 4-hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl, treatment (100 μmol/kg, twice a day) for 10 days prior to the termination of dietary treatment reduced the induction of micronuclei and 8-OHdG in rats fed with a Zn-deficient diet for 6 weeks to levels comparable to those in rats fed with a standard diet for 6 weeks, indicating that superoxide radical participates in the induction of micronuclei and 8-OHdG. In fact, the endogenous superoxide scavenger, Cu/Zn superoxide dismutase, was significantly reduced in the bone marrow cells of rats fed with a Zn-deficient diet for 6 weeks when compared to those of rats fed with a standard diet for 6 weeks (p?<?0.005). These observations demonstrate that Zn deficiency elevates the frequency of micronuclei and the induction of 8-OHdG through an increase in the biological action of the superoxide radical. This suggests an increase in carcinogenic initiation resulting from Zn deficiency-induced oxidative stress.     

The Expression of a Barley HνNAS1 Nicotianamine Synthase Gene Promoter-gus Fusion Gene in Transgenic Tobacco is Induced by Fe-deficiency in Roots

Nicotianamine (NA) is a precursor for mugineic acid-family phytosiderophores, which are a critical component of the Fe aquisition process in graminaceous plants. In addition, nicotianamine synthase (NAS) is strongly induced in these plants by Fe deficiency. NA is essential for Fe metabolism also in dicots, but NAS is not induced by Fe deficiency. We introduced a barley HνNAS1 promoter-gus fusion gene into tobacco. GUS activity was induced in the roots of these plants by Fe deficiency, and was constitutively expressed at a low level in their leaves.     

Variation in Sulfur and Selenium Accumulation Is Controlled by Naturally Occurring Isoforms of the Key Sulfur Assimilation Enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 across the Arabidopsis Species Range

Natural variation allows the investigation of both the fundamental functions of genes and their role in local adaptation. As one of the essential macronutrients, sulfur is vital for plant growth and development and also for crop yield and quality. Selenium and sulfur are assimilated by the same process, and although plants do not require selenium, plant-based selenium is an important source of this essential element for animals. Here, we report the use of linkage mapping in synthetic F2 populations and complementation to investigate the genetic architecture of variation in total leaf sulfur and selenium concentrations in a diverse set of Arabidopsis (Arabidopsis thaliana) accessions. We identify in accessions collected from Sweden and the Czech Republic two variants of the enzyme ADENOSINE 5′-PHOSPHOSULFATE REDUCTASE2 (APR2) with strongly diminished catalytic capacity. APR2 is a key enzyme in both sulfate and selenate reduction, and its reduced activity in the loss-of-function allele apr2-1 and the two Arabidopsis accessions Hodonín and Shahdara leads to a lowering of sulfur flux from sulfate into the reduced sulfur compounds, cysteine and glutathione, and into proteins, concomitant with an increase in the accumulation of sulfate in leaves. We conclude from our observation, and the previously identified weak allele of APR2 from the Shahdara accession collected in Tadjikistan, that the catalytic capacity of APR2 varies by 4 orders of magnitude across the Arabidopsis species range, driving significant differences in sulfur and selenium metabolism. The selective benefit, if any, of this large variation remains to be explored.Sulfur is one of the essential mineral nutrients for all organisms and is required for the biosynthesis of sulfur-containing amino acids, lipids, vitamins, and secondary metabolites, the catalytic and regulatory activity of enzymes, and the stabilization of protein structures. However, animals can only utilize sulfur that has been incorporated into biomolecules primarily originating from plants. Sufficient sulfur fertilization is also important to maximize yields of various crops, including rapeseed (Brassica napus) and wheat (Triticum aestivum; Bloem et al., 2004; Dubousset et al., 2010; Steinfurth et al., 2012). Interestingly, sulfur fertilization can also be important for food quality, with well-fertilized wheat crops producing flour with improved bread-making qualities (Shahsavani and Gholami, 2008).Over the last 50 years, the levels of sulfur in soils have been impacted significantly by the release of sulfur dioxide into the atmosphere as a result of the combustion of fossil fuels (Menz and Seip, 2004). In the atmosphere, sulfur dioxide is oxidized to sulfuric acid and thereafter deposited on soils as acid rain. Regulations controlling the release of sulfur dioxide from power stations introduced in the 1970s have caused significant declines in sulfate deposition due to this acid rain (Menz and Seip, 2004). These reductions in sulfate deposition are significant enough to have increased the need for sulfur fertilization of agricultural crops such as rapeseed and wheat (Dubousset et al., 2010; Steinfurth et al., 2012). The essentiality of sulfur for plants and the relatively recent major fluctuations in soil sulfate deposition make the study of natural variation in sulfur homeostasis by plants attractive. Such studies not only have the potential to identify new molecular mechanisms involved in sulfur homeostasis but also could provide a platform for probing at the genetic level interactions between natural plant populations and a changing soil environment.The sulfur analog selenium is also widely distributed in soil and is incorporated into biomolecules in plants in place of sulfur via sulfur assimilatory processes. Although selenium is not required by plants, it is essential for animals, and plant-based selenium is the primary source of this important nutrient for humans. In animals, selenium plays a vital role in numerous different selenoproteins involved in redox reactions, selenium storage, and hormone biosynthesis (Underwood, 1981; Rayman, 2012; Roman et al., 2014). In these proteins, selenium is incorporated as seleno-Cys. Unlike plants where selenium is incorporated into biomolecules nonspecifically as a sulfur analog, seleno-Cys in animals is biosynthesized by the action of a specific seleno-Cys synthase that converts Ser-tRNA to seleno-Cys-tRNA (Roman et al., 2014). To help prevent the negative health effects of selenium deficiency in the diet, fortification of various foods with selenium is already practiced, and biofortification of crops such as wheat, through the addition of selenium to fertilizers, is being proposed (Lyons et al., 2003). Moreover, there is a growing body of studies suggesting that supraoptimal dietary levels of selenium in the form of seleno-Met and seleno-methylseleno-Cys may be helpful in preventing certain cancers, although the efficacy of these supplements is still debated (Rayman, 2012; Steinfurth et al., 2012). Therefore, the identification of genes that control variation in the uptake and metabolism of sulfur and selenium in plants is not only an essential task for understanding the molecular mechanisms of plant nutrition but also important for crop yield, food quality, and human health.Plants mainly take up sulfur from the soil in the form of sulfate. After uptake of sulfate via the sulfate transporters SULTR1;1 and SULTR1;2 in the root (Yoshimoto et al., 2007; Barberon et al., 2008), sulfate needs to be reduced to sulfide before it can be incorporated into Cys, the first sulfur-containing compound in the assimilatory process (for review, see Takahashi et al., 2011). Sulfate is first activated through adenylation by ATP SULFURYLASE (ATPS) to form adenosine 5′-phosphosulfate (APS) in both plastids and the cytosol (Rotte and Leustek, 2000). After activation, sulfate as APS is reduced to sulfite by APS REDUCTASE (APR) using reduced glutathione (GSH) as the electron donor (Gutierrez-Marcos et al., 1996; Setya et al., 1996). Alternatively, APS can be phosphorylated by APS kinase to form 3′-phosphoadenosine 5′-phosphosulfate (PAPS), which acts as the sulfate donor for sulfotransferases to incorporate sulfate directly into saccharides and secondary metabolites such as glucosinolates (Mugford et al., 2009). Sulfite produced by APR is further reduced to sulfide by sulfite reductase (Khan et al., 2010). In the final step, sulfide is combined with O-acetyl-Ser to form Cys, catalyzed by O-acetyl-Ser(thiol)lyase (Wirtz and Hell, 2006). In plants, selenium is taken up as selenate via sulfate transporters (Shibagaki et al., 2002). After uptake, it is thought that selenate is reduced to selenite via the action of ATP sulfurylase and APS reductase (Shaw and Anderson, 1972; Pilon-Smits et al., 1999; Sors et al., 2005a, 2005b). Selenite is most likely nonenzymatically reduced to selenide and combined with O-acetyl-Ser to form seleno-Cys catalyzed by O-acetyl-Ser(thiol)lyase (Ng and Anderson, 1978).Sulfate uptake and reduction represent the two control points for sulfur homeostasis (Kopriva et al., 2009). Based on sequence similarity, there are 14 genes annotated as sulfate transporters in the Arabidopsis (Arabidopsis thaliana) genome, and at least six of them have evidence supporting their functions (Kopriva et al., 2009; Takahashi, 2010). Among these, SULTR1;1 and SULTR1;2 encode high-affinity sulfate transporters in the root responsible for sulfate uptake from the soil solution (Rouached et al., 2008; Takahashi, 2010). Genes encoding both ATPS and APR also form gene families in Arabidopsis, with ATPS being encoded by four genes and APR by three (Kopriva et al., 2009). ATPS enzymes localize to both the cytosol and plastids, whereas APR enzymes localize solely to the plastids (Lunn et al., 1990; Rotte and Leustek, 2000). ATPS1 and APR2 are responsible for the majority of ATP sulfurylase and APS reductase activity of seedlings.In a quantitative trait locus (QTL) analysis of sulfate content in Arabidopsis leaves using recombinant inbred lines generated by crossing Bayreuth (Bay-0) and Shahdara (Sha), APR2 was identified as a locus controlling variation in sulfate accumulation between the Bay-0 and Sha accessions (Loudet et al., 2007). The causal quantitative trait nucleotide in the Sha allele of APR2 results in a substitution of Ala-399 to Glu-399. The substitution localizes in the thioredoxin active site, which reduces the affinity of APR2 for GSH and results in a loss of 99.8% of enzyme activity. The loss-of-function Sha APR2 allele leads to a significant decrease in sulfate reduction and a concomitant increase in leaf sulfate accumulation. In the same analysis, a second QTL for leaf sulfate accumulation was described (Loudet et al., 2007). This second QTL was recently established to be driven by an ATPS1 expression-level polymorphism between Bay-0 and Sha, with Bay-0 showing reduced expression of ATPS1 compared with Sha (Koprivova et al., 2013).Both Loudet et al. (2007) and Koprivova et al. (2013) focused their studies on the genetic architecture of natural variation for leaf sulfate using a single recombinant population created by crossing the Bay-0 and Sha accessions. This recombinant inbred population has proved a powerful tool for the identification of novel alleles controlling several different traits (Loudet et al., 2008; Jiménez-Gómez et al., 2010; Jasinski et al., 2012; Pineau et al., 2012; Anwer et al., 2014). However, because this set of recombinant inbred lines is composed of genotypes from only two accessions, its value is limited when trying to understand allelic diversity across the Arabidopsis species as a whole. To address this limitation, we performed experiments using a set of 349 Arabidopsis accessions collected from across the species range. These 349 accessions were selected from a worldwide collection of 5,810 accessions sampled to minimize redundancy and close family relatedness (Baxter et al., 2010; Platt et al., 2010). To allow us to further probe Arabidopsis species-wide allelic diversity, we also utilized the complete genome sequences of 855 Arabidopsis accessions from the 1,001 Genomes Project (http://signal.salk.edu/atg1001/index.php).Since sulfate uptake and accumulation is only one step in the complex process of sulfur assimilation in plants, uncovering the genetic architecture of sulfate accumulation, as performed by Loudet et al. (2007) and Koprivova et al. (2013), could potentially overlook genetic variation in other important aspects of sulfur metabolism. To enable us to identify natural genetic variation in sulfur homeostasis beyond just sulfate accumulation, we chose to quantify total leaf sulfur. This potentially allowed us to capture variation in the accumulation of both sulfate and other important sulfur-containing metabolites such as GSH and glucosinolates.Furthermore, to test genetically the long-held assumption that selenium in plants is metabolized as a sulfur analog, we also phenotyped the same set of plants for total leaf selenium. Through a combination of high-throughput elemental analysis, linkage mapping, genetic and transgenic complementation, reciprocal grafting, and protein haplotype analysis, we have established that the natural variation in both leaf sulfur and selenium content in Arabidopsis is controlled by several rare APR2 variants across the whole of the Arabidopsis species.     

3′-Modified oligodeoxyribonucleotides for the study of 2-deoxyribose damage in DNA

Well-defined substrates for the study of oxidative processes are important for the elucidation of the role of DNA damage in the etiology of diseases such as cancer. We have synthesized 3′-modified oligodeoxyribonucleotides (ODNs) using 5′ → 3′ ‘reverse’ DNA synthesis for the study of 2-deoxyribose oxidative damage to DNA. The modified monomers designed for these studies all share a common feature, they lack the naturally occurring 3′-hydroxyl group found in 2-deoxyribonucleosides. Modified H-phosphonates containing 3′-phenyl selenides as well as saturated and unsaturated sugars were obtained and incorporated in ODNs. These ODNs were used to investigate the fate of C3′-dideoxyribonucleotide radicals in DNA.     

Simian Virus 40 Mutants with Deletions at the 3′ End of the Early Region Are Defective in Adenovirus Helper Function

Coinfection of monkey cells with simian virus 40 (SV40) and adenovirus type 2 (Ad2) increased the Ad2 yield 1,000-fold over that obtained by Ad2 infection alone of monkey cells (A. S. Rabson, G. T. O'Conor, I. K. Berezesky, and F. J. Paul, Proc. Soc. Exp. Biol. Med. 116:187-190, 1964). The ability of viable mutants of SV40 that contain deletions at various sites in the viral DNA to enhance Ad2 growth in monkey cells was examined. Only those mutants with deletions near the 3' end of the early region were deficient in providing this helper function. Mutants dl1265, lacking 39 base pairs at map position 0.18, and dl1263, lacking 33 base pairs at map position 0.20 (H. van Heuverswyn, C. Cole, P. Berg, and W. Fiers, J. Virol. 30:936-941, 1979), were approximately 4 and 30% as effective as wild-type SV40, respectively. The extent of enhancement of Ad2 yield depended on the multiplicity of infection by SV40, but not by Ad2 (at a multiplicity of infection of 相似文献