Browsing by Author "Tekpinar, Ayten Dizkirici"

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Communication Between Dna Polymerases and Replication Protein a Within the Archaeal Replisome
(Nature Portfolio, 2024) Martinez Carranza, Markel; Vialle, Lea; Madru, Clement; Cordier, Florence; Tekpinar, Ayten Dizkirici; Haouz, Ahmed; Sauguet, Ludovic
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 demonstrate 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 unveil 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 is 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 central replication factors such as RPA, which regulate genome maintenance in Archaea and Eukaryotes.
Fast and Efficient Purification of Sars-Cov Rna Dependent Rna Polymerase Complex Expressed in Escherichia Coli
(Public Library Science, 2021) Madru, Clement; Tekpinar, Ayten Dizkirici; Rosario, Sandrine; Czernecki, Dariusz; Brule, Sebastien; Sauguet, Ludovic; Delarue, Marc
To 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 similar to 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).
Fungal Systematics and Evolution: Fuse 10
(verlag Ferdinand Berger Sohne Gesellschaft Mbh, 2024) Lambert, Herman; Acar, Ismail; Tekpinar, Ayten Dizkirici; Fortin, Guy; Ivanushkina, Nataliya; Jacobs, Karin; Haelewaters, Danny
In this 10th contribution to the Fungal Systematics and Evolution series published by Sydowia, 14 species are formally described: Amanita cingulatoides from Canada, A. confundens, A. elongatior, A. fusca, A. fuscozonata, A. leptorhacopus, A. magna, A. olivaceodisca, A. piceina, A. pulla, and A. quercifulva from Canada and the USA, Hebeloma uzunii from T & uuml;rkiye, Pleurotus overstrandensis from South Africa, and Saksenaea ozerskayae from Russia. Pluteus keselakii is reported for the first time from Czechia and Slovenia. Three invalidly published names are validated with corrected typification: Cortinarius balteatoindicus, C. indopurpurascens, and C. ulkhagarhiensis. Finally, Thaxterogaster shoreae is combined into Cortinarius.
Phylogeography and Phylogeny of Genus Quercus L. (Fagaceae) in Turkey Implied by Variations of Trnt(ugu)-L(uaa)< (Gaa) Chloroplast Dna Region
(Springer Heidelberg, 2021) Tekpinar, Ayten Dizkirici; Aktas, Caner; Kansu, Cigdem; Duman, Hayri; Kaya, Zeki
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 h(T) > v(T) 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.
Utility of Various Molecular Markers in Fungal Identification and Phylogeny
(Gebruder Borntraeger, 2019) Tekpinar, Ayten Dizkirici; Kalmer, Aysenur
Identification of fungi to the species level has some challenges because of great diversity in morphology and ecology. Therefore, developing an efficient species recognition system which is applicable for all fungi seems difficult. Currently, DNA sequences of different regions have become powerful molecular data for rapid identification of specimens and overcoming the different sets of traditional criteria used for describing fungal species. However, deciding the potential region(s) as DNA barcode is a crucial step to identify biological specimens and to assign them to a given species. In the present review, usefulness of several DNA markers (ITS, LSU, SSU, COX1, RPB1, RPB2, beta-tubulin, MCM7, TEF1-alpha, gamma-actin, ATP6 and CaM genes) were investigated based on current studies. The pros and cons of each gene in Ascomycota, Basidiomycota and other fungal phyla were discussed. Even though numerous studies were completed to decide a marker as DNA barcode for a specific taxon it was observed that potential DNA barcodes vary from species to species. However, ITS was indicated by lots of studies as the potential barcode for most of fungi due to high degree of interspecific variability, conserved primer sites and multiple copy nature in the genome. Several protein-coding regions such as TEF1-alpha, RPB1, RPB2, beta-tubulin and CaM genes may be accepted as secondary barcodes for the vast majority of Ascomycota genera such as Penicillium, Aspergillus and Fusarium. Therefore, concatenated alignment of the ITS region with one or more protein-coding genes may be effective for finer-scale species-level identification of specific fungi.