Radiocarbon Chronologies and Extinction Dynamics of the Late Quaternary Mammalian Megafauna of the Taimyr Peninsula, Russian Federation

This paper presents 75 new radiocarbon dates based on late Quaternary mammal remains recovered from eastern Taimyr Peninsula and adjacent parts of the northern Siberian lowlands, Russian Federation, including specimens of woolly mammoth (Mammuthus primigenius), steppe bison (Bison priscus), muskox (Ovibos moschatus), moose (Alces alces), reindeer (Rangifer tarandus), horse (Equus caballus) and wolf (Canis lupus). New evidence permits reanalysis of megafaunal extinction dynamics in the Asian high Arctic periphery. Increasingly, radiometric records of individual species show evidence of a gap at or near the Pleistocene/Holocene boundary (PHB). In the past, the PHB gap was regarded as significant only when actually terminal, i.e., when it marked the apparent ‘‘last’’ occurrence of a species (e.g., current ‘‘last’’ occurrence date for woolly mammoth in mainland Eurasia is 9600 yr BP). However, for high Arctic populations of horses and muskoxen the gap marks an interruption rather than extinction, because their radiocarbon records resume, nearly simultaneously, much later in the Holocene. Taphonomic effects, ΔC14 flux, and biased sampling are unlikely explanations for these hiatuses. A possible explanation is that the gap is the signature of an event, of unknown nature, that prompted the nearly simultaneous crash of many megafaunal populations in the high Arctic and possibly elsewhere in Eurasia.

Novel Binding Mode of a Potent and Selective Tankyrase Inhibitor

Tankyrases (TNKS1 and TNKS2) are key regulators of cellular processes such as telomere pathway and Wnt signaling. IWRs (inhibitors of Wnt response) have recently been identified as potent and selective inhibitors of tankyrases. However, it is not clear how these IWRs interact with tankyrases. Here we report the crystal structure of the catalytic domain of human TNKS1 in complex with IWR2, which reveals a novel binding site for tankyrase inhibitors. The TNKS1/IWR2 complex provides a molecular basis for their strong and specific interactions and suggests clues for further development of tankyrase inhibitors

Uracil DNA N-Glycosylase Promotes Assembly of Human Centromere Protein A

Uracil is removed from DNA by the conserved enzyme Uracil DNA N-glycosylase (UNG). Previously, we observed that inhibiting UNG in Xenopus egg extracts blocked assembly of CENP-A, a histone H3 variant. CENP-A is an essential protein in all species, since it is required for chromosome segregation during mitosis. Thus, the implication of UNG in CENP-A assembly implies that UNG would also be essential, but UNG mutants lacking catalytic activity are viable in all species. In this paper, we present evidence that UNG2 colocalizes with CENP-A and H2AX phosphorylation at centromeres in normally cycling cells. Reduction of UNG2 in human cells blocks CENP-A assembly, and results in reduced cell proliferation, associated with increased frequencies of mitotic abnormalities and rapid cell death. Overexpression of UNG2 induces high levels of CENP-A assembly in human cells. Using a multiphoton laser approach, we demonstrate that UNG2 is rapidly recruited to sites of DNA damage. Taken together, our data are consistent with a model in which the N-terminus of UNG2 interacts with the active site of the enzyme and with chromatin

Identification and Characterization of Inhibitors of Human Apurinic/apyrimidinic Endonuclease APE1

APE1 is the major nuclease for excising abasic (AP) sites and particular 3′-obstructive termini from DNA, and is an integral participant in the base excision repair (BER) pathway. BER capacity plays a prominent role in dictating responsiveness to agents that generate oxidative or alkylation DNA damage, as well as certain chain-terminating nucleoside analogs and 5-fluorouracil. We describe within the development of a robust, 1536-well automated screening assay that employs a deoxyoligonucleotide substrate operating in the red-shifted fluorescence spectral region to identify APE1 endonuclease inhibitors. This AP site incision assay was used in a titration-based high-throughput screen of the Library of Pharmacologically Active Compounds (LOPAC1280), a collection of well-characterized, drug-like molecules representing all major target classes. Prioritized hits were authenticated and characterized via two high-throughput screening assays – a Thiazole Orange fluorophore-DNA displacement test and an E. coli endonuclease IV counterscreen – and a conventional, gel-based radiotracer incision assay. The top, validated compounds, i.e. 6-hydroxy-DL-DOPA, Reactive Blue 2 and myricetin, were shown to inhibit AP site cleavage activity of whole cell protein extracts from HEK 293T and HeLa cell lines, and to enhance the cytotoxic and genotoxic potency of the alkylating agent methylmethane sulfonate. The studies herein report on the identification of novel, small molecule APE1-targeted bioactive inhibitor probes, which represent initial chemotypes towards the development of potential pharmaceuticals

Identification of four families of yCCR4- and Mg(2+)-dependent endonuclease-related proteins in higher eukaryotes, and characterization of orthologs of yCCR4 with a conserved leucine-rich repeat essential for hCAF1/hPOP2 binding

BACKGROUND: The yeast yCCR4 factor belongs to the CCR4-NOT transcriptional regulatory complex, in which it interacts, through its leucine-rich repeat (LRR) motif with yPOP2. Recently, yCCR4 was shown to be a component of the major cytoplasmic mRNA deadenylase complex, and to contain a fold related to the Mg(2+)-dependent endonuclease core. RESULTS: Here, we report the identification of nineteen yCCR4-related proteins in eukaryotes (including yeast, plants and animals), which all contain the yCCR4 endonuclease-like fold, with highly conserved CCR4-specific residues. Phylogenetic and genomic analyses show that they form four distinct families, one of which contains the yCCR4 orthologs. The orthologs in animals possess a leucine-rich repeat domain. We show, using two-hybrid and far-Western assays, that the human member binds to the human yPOP2 homologs, i.e. hCAF1 and hPOP2, in a LRR-dependent manner. CONCLUSIONS: We have identified the mammalian orthologs of yCCR4 and have shown that the human member binds to the human yPOP2 homologs, thus strongly suggesting conservation of the CCR4-NOT complex from yeast to human. All members of the four identified yCCR4-related protein families show stricking conservation of the endonuclease-like catalytic motifs of the yCCR4 C-terminal domain and therefore constitute a new family of potential deadenylases in mammals

An AP Endonuclease 1–DNA Polymerase β Complex: Theoretical Prediction of Interacting Surfaces

Abasic (AP) sites in DNA arise through both endogenous and exogenous mechanisms. Since AP sites can prevent replication and transcription, the cell contains systems for their identification and repair. AP endonuclease (APEX1) cleaves the phosphodiester backbone 5′ to the AP site. The cleavage, a key step in the base excision repair pathway, is followed by nucleotide insertion and removal of the downstream deoxyribose moiety, performed most often by DNA polymerase beta (pol-β). While yeast two-hybrid studies and electrophoretic mobility shift assays provide evidence for interaction of APEX1 and pol-β, the specifics remain obscure. We describe a theoretical study designed to predict detailed interacting surfaces between APEX1 and pol-β based on published co-crystal structures of each enzyme bound to DNA. Several potentially interacting complexes were identified by sliding the protein molecules along DNA: two with pol-β located downstream of APEX1 (3′ to the damaged site) and three with pol-β located upstream of APEX1 (5′ to the damaged site). Molecular dynamics (MD) simulations, ensuring geometrical complementarity of interfaces, enabled us to predict interacting residues and calculate binding energies, which in two cases were sufficient (∼−10.0 kcal/mol) to form a stable complex and in one case a weakly interacting complex. Analysis of interface behavior during MD simulation and visual inspection of interfaces allowed us to conclude that complexes with pol-β at the 3′-side of APEX1 are those most likely to occur in vivo. Additional multiple sequence analyses of APEX1 and pol-β in related organisms identified a set of correlated mutations of specific residues at the predicted interfaces. Based on these results, we propose that pol-β in the open or closed conformation interacts and makes a stable interface with APEX1 bound to a cleaved abasic site on the 3′ side. The method described here can be used for analysis in any DNA-metabolizing pathway where weak interactions are the principal mode of cross-talk among participants and co-crystal structures of the individual components are available

Characterization of magnesium requirement of human 5'-tyrosyl DNA phosphodiesterase mediated reaction

<p>Abstract</p> <p>Background</p> <p>Topo-poisons can produce an enzyme-DNA complex linked by a 3'- or 5'-phosphotyrosyl covalent bond. 3'-phosphotyrosyl bonds can be repaired by tyrosyl DNA phosphodiesterase-1 (TDP1), an enzyme known for years, but a complementary human enzyme 5'-tyrosyl DNA phosphodiesterase (hTDP2) that cleaves 5'-phosphotyrosyl bonds has been reported only recently. Although hTDP2 possesses both 3'- and 5'- tyrosyl DNA phosphodiesterase activity, the role of Mg²⁺in its activity was not studied in sufficient details.</p> <p>Results</p> <p>In this study we showed that purified hTDP2 does not exhibit any 5'-phosphotyrosyl phosphodiesterase activity in the absence of Mg²⁺/Mn²⁺, and that neither Zn²⁺or nor Ca²⁺can activate hTDP2. Mg²⁺also controls 3'-phosphotyrosyl activity of TDP2. In MCF-7 cell extracts and de-yolked zebrafish embryo extracts, Mg²⁺controlled 5'-phosphotyrosyl activity. This study also showed that there is an optimal Mg²⁺concentration above which it is inhibitory for hTDP2 activity.</p> <p>Conclusion</p> <p>These results altogether reveal the optimal Mg²⁺requirement in hTDP2 mediated reaction.</p

Crystal Structures of the FAK Kinase in Complex with TAE226 and Related Bis-Anilino Pyrimidine Inhibitors Reveal a Helical DFG Conformation

Focal Adhesion Kinase (FAK) is a non-receptor tyrosine kinase required for cell migration, proliferation and survival. FAK overexpression has been documented in diverse human cancers and is associated with a poor clinical outcome. Recently, a novel bis-anilino pyrimidine inhibitor, TAE226, was reported to efficiently inhibit FAK signaling, arrest tumor growth and invasion and prolong the life of mice with glioma or ovarian tumor implants. Here we describe the crystal structures of the FAK kinase bound to TAE226 and three related bis-anilino pyrimidine compounds. TAE226 induces a conformation of the N-terminal portion of the kinase activation loop that is only observed in FAK, but is distinct from the conformation in both the active and inactive states of the kinase. This conformation appears to require a glycine immediately N-terminal to the “DFG motif”, which adopts a helical conformation stabilized by interactions with TAE226. The presence of a glycine residue in this position contributes to the specificity of TAE226 and related compounds for FAK. Our work highlights the fact that kinases can access conformational space that is not necessarily utilized for their native catalytic regulation, and that such conformations can explain and be exploited for inhibitor specificity

Validation of Plasmodium falciparum dUTPase as the target of 5'-tritylated deoxyuridine analogues with anti-malarial activity

BACKGROUND: Malaria remains as a major global problem, being one of the infectious diseases that engender highest mortality across the world. Due to the appearance of resistance and the lack of an effective vaccine, the search of novel anti-malarials is required. Deoxyuridine 5'-triphosphate nucleotido-hydrolase (dUTPase) is responsible for the hydrolysis of dUTP to dUMP within the parasite and has been proposed as an essential step in pyrimidine metabolism by providing dUMP for thymidylate biosynthesis. In this work, efforts to validate dUTPase as a drug target in Plasmodium falciparum are reported. METHODS: To investigate the role of PfdUTPase in cell survival different strategies to generate knockout mutants were used. For validation of PfdUTPase as the intracellular target of four inhibitors of the enzyme, mutants overexpressing PfdUTPase and HsdUTPase were created and the IC50 for each cell line with each compound was determined. The effect of these compounds on dUTP and dTTP levels from P. falciparum was measured using a DNA polymerase assay. Detailed localization studies by indirect immunofluorescence microscopy and live cell imaging were also performed using a cell line overexpressing a Pfdut-GFP fusion protein. RESULTS:Different attempts of disruption of the dut gene of P. falciparum were unsuccessful while a 3' replacement construct could recombine correctly in the locus suggesting that the enzyme is essential. The four 5'-tritylated deoxyuridine analogues described are potent inhibitors of the P. falciparum dUTPase and exhibit antiplasmodial activity. Overexpression of the Plasmodium and human enzymes conferred resistance against selective compounds, providing chemical validation of the target and confirming that indeed dUTPase inhibition is involved in anti-malarial activity. In addition, incubation with these inhibitors was associated with a depletion of the dTTP pool corroborating the central role of dUTPase in dTTP synthesis. PfdUTPase is mainly localized in the cytosol. CONCLUSION: These results strongly confirm the pivotal and essential role of dUTPase in pyrimidine biosynthesis of P. falciparum intraerythrocytic stages

Predicting Inactive Conformations of Protein Kinases Using Active Structures: Conformational Selection of Type-II Inhibitors

Protein kinases have been found to possess two characteristic conformations in their activation-loops: the active DFG-in conformation and the inactive DFG-out conformation. Recently, it has been very interesting to develop type-II inhibitors which target the DFG-out conformation and are more specific than the type-I inhibitors binding to the active DFG-in conformation. However, solving crystal structures of kinases with the DFG-out conformation remains a challenge, and this seriously hampers the application of the structure-based approaches in development of novel type-II inhibitors. To overcome this limitation, here we present a computational approach for predicting the DFG-out inactive conformation using the DFG-in active structures, and develop related conformational selection protocols for the uses of the predicted DFG-out models in the binding pose prediction and virtual screening of type-II ligands. With the DFG-out models, we predicted the binding poses for known type-II inhibitors, and the results were found in good agreement with the X-ray crystal structures. We also tested the abilities of the DFG-out models to recognize their specific type-II inhibitors by screening a database of small molecules. The AUC (area under curve) results indicated that the predicted DFG-out models were selective toward their specific type-II inhibitors. Therefore, the computational approach and protocols presented in this study are very promising for the structure-based design and screening of novel type-II kinase inhibitors