Phylogeography and phylogeny of genus Quercus L. (Fagaceae) in Turkey implied by variations of trnT (UGU) -L (UAA) -F (GAA) chloroplast DNA region

The genus Quercus L. is one of the most abundant and important genera of woody plants in the Northern Hemisphere as well as in Turkey. In the current study which is the most comprehensive study dealing with Turkish oaks, sequence variations of three noncoding regions (trnT(UGU)-L(UAA) IGS, trnL(UAA)intron, trnL(UAA)-F(GAA) IGS) of chloroplast DNA (cpDNA) were used for phylogeographic and phylogenetic analysis on 319 individuals representing 23 taxa (17 species). The trnT(UGU)-L(UAA) region was found to be the most variable and parsimony informative region. Twenty-eight cpDNA haplotypes were identified based on 34 substitutions and 22 indels. High number of haplotypes and hT > vT observed in populations of oaks in Turkey indicated that the Anatolian Peninsula might have been a refugium at Glacial Periods. Phylogeographic construction and molecular variance analysis revealed that Quercus cpDNA haplotypes were geographically structured. Although local haplotype sharing among species from same infrageneric clades was common, levels of hybridization differ between species pairs. Haplotype analysis revealed four infrageneric clades, namely Section Quercus, Section Cerris and two clades corresponding to Section Ilex, namely “Ilex” and “Coccifera.” Furthermore, a Section Cerris haplotype was detected in the Aegean members of Q. ilex and Q. coccifera. Section Ponticae was placed in the Section Quercus cluster. In contrast to the phylogenetic reconstructions based on the nuclear DNA sequence data, Group Ilex seems to be polyphyletic based on plastome phylogeny. Chloroplast phylogeny of oaks reflects the traces of recent and ancient introgression events during diversification of species. In addition to this, incomplete linkage sorting may also explain this polymorphic assemblage. Therefore, further investigation is required to clarify the cpDNA phylogeny of oaks, especially for Section Ilex

Phylogenetic relationships between Oxytropis DC. and Astragalus L. species native to an Old World diversity center inferred from nuclear ribosomal ITS and plastid matK gene sequences

Oxytropis and Astragalus represent one of the largest angiosperm genera complexes. Although phylogenetic studies of this complex exist, the evolutionary relationships among Astragalus and Oxytropis species sharing similar habitats in the Old World have not been studied in detail. The phylogenetic relationships among 13 Oxytropis and 56 Astragalus species native to Turkey were inferred from nucleotide sequence variations in the nuclear ribosomal internal transcribed spacer (ITS) and chloroplast maturase-like protein (matK) gene regions. In addition to our samples, 36 Oxytropis ITS and 6 Oxytropis matK sequences were retrieved from GenBank and included in the analysis. Phylogenies derived from a maximum likelihood analysis of the sequences indicated that Oxytropis and Astragalus genera are more likely monophyletic. However, the results suggest that New World Oxytropis species did not evolve by a single adaptive radiation in the genus, but rather from different Old World lineages. The genetic divergence between genera was less when the matK region was analyzed. Although the Oxytropis species did not show high genetic diversity, one subcluster of the genus was always distinctly separated in both trees. This subcluster was formed by the species Oxytropis engizekensis Duman \& Vural and O. persica Boiss., which are also regarded as synonyms in regard to several morphological characters of the genus

Fast and efficient purification of SARS-CoV-2 RNA dependent RNA polymerase complex expressed in Escherichia coli

International audienceTo stop the COVID-19 pandemic due to the Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2), which caused more than 2.5 million deaths to date, new antiviral molecules are urgently needed. The replication of SARS-CoV-2 requires the RNA-dependent RNA polymerase (RdRp), making RdRp an excellent target for antiviral agents. RdRp is a multi-subunit complex composed of 3 viral proteins named nsp7, nsp8 and nsp12 that ensure the ~30 kb RNA genome's transcription and replication. The main strategies employed so far for the overproduction of RdRp consist of expressing and purifying the three subunits separately before assembling the complex in vitro. However, nsp12 shows limited solubility in bacterial expression systems and is often produced in insect cells. Here, we describe an alternative strategy to co-express the full SARS-CoV-2 RdRp in E. coli, using a single plasmid. Characterization of the purified recombinant SARS-CoV-2 RdRp shows that it forms a complex with the expected (nsp7)(nsp8)2(nsp12) stoichiometry. RNA polymerization activity was measured using primer-extension assays showing that the purified enzyme is functional. The purification protocol can be achieved in one single day, surpassing in speed all other published protocols. Our construct is ideally suited for screening RdRp and its variants against very large chemical compounds libraries and has been made available to the scientific community through the Addgene plasmid depository (Addgene ID: 165451)

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.</div

Communication between DNA polymerases and Replication Protein A within the archaeal replisome

Replication Protein A (RPA) plays a pivotal role in DNA replication by coating and protecting exposed single-stranded DNA, and acting as a molecular hub that recruits additional replication factors. We demonstrated that archaeal RPA hosts a winged-helix domain (WH) that interacts with two key actors of the replisome: the DNA primase (PriSL) and the replicative DNA polymerase (PolD). Using an integrative structural biology approach, combining nuclear magnetic resonance, X-ray crystallography and cryo-electron microscopy, we unveiled how RPA interacts with PriSL and PolD through two distinct surfaces of the WH domain: an evolutionarily conserved interface and a novel binding site. Finally, RPA was shown to stimulate the activity of PriSL in a WH-dependent manner. This study provides a molecular understanding of the WH-mediated regulatory activity in conserved central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.</div