Adjacent Phosphate Research Articles

DNA Replication across α-l-(3'-2')-Threofuranosyl Nucleotides Mediated by Human DNA Polymerase η.

α-l-(3'-2')-Threofuranosyl nucleic acid (TNA) pairs with itself, cross-pairs with DNA and RNA, and shows promise as a tool in synthetic genetics, diagnostics, and oligonucleotide therapeutics. We studied in vitro primer insertion and extension reactions catalyzed by human trans-lesion synthesis (TLS) DNA polymerase η (hPol η) opposite a TNA-modified template strand without and in combination with O4-alkyl thymine lesions. Across TNA-T (tT), hPol η inserted mostly dAMP and dGMP, dTMP and dCMP with lower efficiencies, followed by extension of the primer to a full-length product. hPol η inserted dAMP opposite O4-methyl and -ethyl analogs of tT, albeit with reduced efficiencies relative to tT. Crystal structures of ternary hPol η complexes with template tT and O4-methyl tT at the insertion and extension stages demonstrated that the shorter backbone and different connectivity of TNA compared to DNA (3' → 2' versus 5' → 3', respectively) result in local differences in sugar orientations, adjacent phosphate spacings, and directions of glycosidic bonds. The 3'-OH of the primer's terminal thymine was positioned at 3.4 Å on average from the α-phosphate of the incoming dNTP, consistent with insertion opposite and extension past the TNA residue by hPol η. Conversely, the crystal structure of a ternary hPol η·DNA·tTTP complex revealed that the primer's terminal 3'-OH was too distant from the tTTP α-phosphate, consistent with the inability of the polymerase to incorporate TNA. Overall, our study provides a better understanding of the tolerance of a TLS DNA polymerase vis-à-vis unnatural nucleotides in the template and as the incoming nucleoside triphosphate.

Distribution of trace and rare earth elements in the estuary of Ría de Huelva (SW Spain): Field data and geochemical modeling

The geochemistry of the Ría de Huelva estuary (Spain) has been widely studied owing to its unique conditions due to mixing of acid mine drainage (AMD), river water and seawater, which have been aggravated by the existence of an adjacent phosphate fertilizer plant and its associated phosphogypsum stack.To better understand the complex geochemistry of the overall estuarine system, we make use of (1) measured concentrations of relevant trace elements (rare earth elements (REEs) and metal(loid)s) in the sediments and waters of the estuary and (2) geochemical modeling which combines mixing and adsorption processes.Our study sheds light on the elemental distribution in the sediments and water in the water-mixing area. As seawater neutralizes the acidity of the river water (pH increases to 6.2) colloids of Fe- and Al-oxyhydroxysulphates (schwertmannite and basaluminite, respectively) precipitate as concentrations of aqueous Fe and Al increase. These phases have a high adsorption capacity and play a critical role in the geochemistry of the aquatic system by retaining Cu, Pb, Cr, As, P, REEs and smaller amounts of Zn, Co, Ni and Mn. The geochemical model reproduces the behaviour of the aqueous REEs, requiring high S/L ratios at pH ≥ 3.5 and participation of sediments and colloids in the reactions to match the field data.The evaluation of the effect of the adjacent phosphogypsum stack on the estuarine geochemistry shows that the stack has a notorious impact on the geochemistry of the surrounding estuarine environment owing to the release of high quantities of phosphate.

Catalytic Metal Ion-Substrate Coordination during Nonenzymatic RNA Primer Extension.

Nonenzymatic template-directed RNA copying requires catalysis by divalent metal ions. The primer extension reaction involves the attack of the primer 3'-hydroxyl on the adjacent phosphate of a 5'-5'-imidazolium-bridged dinucleotide substrate. However, the nature of the interaction of the catalytic metal ion with the reaction center remains unclear. To explore the coordination of the catalytic metal ion with the imidazolium-bridged dinucleotide substrate, we examined catalysis by oxophilic and thiophilic metal ions with both diastereomers of phosphorothioate-modified substrates. We show that Mg2+ and Cd2+ exhibit opposite preferences for the two phosphorothioate substrate diastereomers, indicating a stereospecific interaction of the divalent cation with one of the nonbridging phosphorus substituents. High-resolution X-ray crystal structures of the products of primer extension with phosphorothioate substrates reveal the absolute stereochemistry of this interaction and indicate that catalysis by Mg2+ involves inner-sphere coordination with the nonbridging phosphate oxygen in the pro-SP position, while thiophilic cadmium ions interact with sulfur in the same position, as in one of the two phosphorothioate substrates. These results collectively suggest that during nonenzymatic RNA primer extension with a 5'-5'-imidazolium-bridged dinucleotide substrate the interaction of the catalytic Mg2+ ion with the pro-SP oxygen of the reactive phosphate plays a crucial role in the metal-catalyzed SN2(P) reaction.

Simultaneous Solar-Thermal Desalination and Catalytic Degradation of Wastewater Containing Both Salt Ions and Organic Contaminants.

Although solar steam generation is promising in generating clean water by desalinating seawater, it is powerless to totally degrade organic contaminants in the seawater. Herein, solar steam generation and catalytic degradation are integrated to generate clean water by simultaneous solar-driven desalination and catalytic degradation of wastewater containing both salt ions and organic contaminants. Stepwise decoration of three-dimensional nickel foam with polypyrrole, reduced graphene oxide (RGO), and cobalt phosphate is realized to obtain polypyrrole/RGO/cobalt phosphate/nickel foam (PGCN) hybrids for solar-driven desalination and catalytic degradation of wastewater containing antibiotics and salt ions. The oxygen-containing groups of the RGO integrated with the porous nickel foam make the porous PGCN hybrid hydrophilic and ensure the upward transport of water to the evaporation surface, and the oxygen vacancies of the cobalt phosphate allow the PGCN to generate abundant highly active singlet oxygen that could still exhibit excellent catalytic degradation performances in the high salinity and highly alkaline environment of seawater. In addition to the high solar light absorbance and satisfactory solar-thermal conversion efficiency of polypyrrole and RGO, the thermally conductive nickel foam skeleton can effectively transfer the heat generated by the solar-thermal energy conversion to the adjacent cobalt phosphate catalyst and nearby wastewater, achieving a solar-thermal-promoted catalytic degradation of organic contaminants. Therefore, a high pure water evaporation rate of 2.08 kg m-2 h-1 under 1 sun irradiation and 100% catalytic degradation of Norfloxacin and dyes are achieved. The PGCN hybrid is highly efficient in purifying seawater containing 10 ppm Norfloxacin and simultaneously achieves a high purification efficiency of 100 kg m-2 h-1.

The caesium phosphates Cs3(H1.5PO4)2(H2O)2, Cs3(H1.5PO4)2, Cs4P2O7(H2O)4, and CsPO3

The caesium phosphates Cs3(H1.5PO4)2(H2O)2 and Cs3(H1.5PO4)2 were obtained from aqueous solutions, and Cs4P2O7(H2O)4 and CsPO3 from solid state reactions, respectively. Cs3(H1.5PO4)2, Cs4P2O7(H2O)4, and CsPO3 were fully structurally characterized for the first time on basis of single-crystal X-ray diffraction data recorded at − 173 °C. Monoclinic Cs3(H1.5PO4)2 (Z = 2, C2/m) represents a new structure type and comprises hydrogen phosphate groups involved in the formation of a strong non-symmetrical hydrogen bond (accompanied by a disordered H atom over a twofold rotation axis) and a very strong symmetric hydrogen bond (with the H atom situated on an inversion centre) with symmetry-related neighbouring anions. Triclinic Cs4P2O7(H2O)4 (Z = 2, Pbar{1}) crystallizes also in a new structure type and is represented by a diphosphate group with a P–O–P bridging angle of 128.5°. Although H atoms of the water molecules were not modelled, O···O distances point to hydrogen bonds of medium strengths in the crystal structure. CsPO3 is monoclinic (Z = 4, P21/n) and belongs to the family of catena-polyphosphates (MPO3)n with a repetition period of 2. It is isotypic with the room-temperature modification of RbPO3. The crystal structure of Cs3(H1.5PO4)2(H2O)2 was re-evaluated on the basis of single-crystal X-ray diffraction data at − 173 °C, revealing that two adjacent hydrogen phosphate anions are connected by a very strong and non-symmetrical hydrogen bond, in contrast to the previously described symmetrical bonding situation derived from room temperature X-ray diffraction data. In the four title crystal structures, coordination numbers of the caesium cations range from 7 to 12.Graphic abstract

WAPHCO Project: How to Produce High-Quality Merchant Grade Acid from El Wady Phosphate Rock Charged with Impurities?

The WAPHCO Project consists of the construction of a phosphoric acid plant producing 500,000 metric tons of P2O5 per year as merchant grade acid (“MGA”) from the adjacent Abu Tartour phosphate ore mine in the southwestern desert of Egypt. The main issue with that ore is the high content of minor elements like iron, aluminum and magnesium. From a chemical standpoint, those elements lead to the formation of gels impeding negatively the production of phosphoric acid. During a preliminary bench scale test performed by Prayon, the acid could not be concentrated above 42% P2O5 while MGA specification requires at least 52%. At that stage, K-Technologies, Inc. (K-Tech) was introduced into the project to solve those “minor elements” issues. Indeed, the American company has developed and patented a technology to remove the minor element cations from a phos acid solution by continuous ion-exchange. Preliminary bench scale tests were performed successfully by K-Tech on phosphoric acid produced by Prayon from the Egyptian Rock. Consequently, WAPHCO decided to hire Prayon and K-Tech to design the plant based on a combined and integrated pilot plant test work program. Results of the pilot tests were even better than expected as the quality of the produced acid largely exceeded the usual requirement from fertilizer producers in terms of purity degree and post-precipitation characteristics. Moreover, performances of the Prayon Dihydrate Process regarding P2O5 recovery through attack and filtration were enhanced during pilot test phase. The final plant design elaborated after the tests is the consequence of close collaboration between Prayon and K-Tech. Synergies between processes will allow the client to save money on capital and operational costs of the plant, as well as produce a premium product for the marketplace.

Theoretical investigation of the effect of phosphate doping on the aggregation of Au atoms on an Al2O3 (0001) surface

Using density functional theory, the adsorption of Au atoms and Au dimers onto α-Al2O3 (0001) surfaces with various phosphate coverages was investigated. When the phosphate coverage is low (0% or 25%), Au dimers are more stable than isolated Au atoms on the surfaces, and therefore, aggregation of Au atoms into Au dimers is exothermic. In contrast, when the phosphate coverage is high (50% or 100%), isolated Au atoms are more stable than Au dimers on the surface, and Au atom aggregation is endothermic. This difference is caused by differences in the distance between adjacent phosphate groups; at certain distances, the phosphate groups are capable of bridging Au atoms. The Au-Au bond of a Au dimer at a phosphate-phosphate bridge site is destabilized by hole transfer from the Al2O3 surfaces to the bonding σ orbital of the Au dimer. Isolated phosphate groups cannot cause the destabilization. These results provide a good explanation for the mechanism of the suppression of aggregation of Au particles caused by phosphate doping on an Al2O3 surface, and show the importance of charge transfer from a surface to Au atoms for suppressing Au sintering.

Structural basis for the synergy of 4'- and 2'-modifications on siRNA nuclease resistance, thermal stability and RNAi activity.

Chemical modification is a prerequisite of oligonucleotide therapeutics for improved metabolic stability, uptake and activity, irrespective of their mode of action, i.e. antisense, RNAi or aptamer. Phosphate moiety and ribose C2′/O2′ atoms are the most common sites for modification. Compared to 2′-O-substituents, ribose 4′-C-substituents lie in proximity of both the 3′- and 5′-adjacent phosphates. To investigate potentially beneficial effects on nuclease resistance we combined 2′-F and 2′-OMe with 4′-Cα- and 4′-Cβ-OMe, and 2′-F with 4′-Cα-methyl modification. The α- and β-epimers of 4′-C-OMe-uridine and the α-epimer of 4′-C-Me-uridine monomers were synthesized and incorporated into siRNAs. The 4′α-epimers affect thermal stability only minimally and show increased nuclease stability irrespective of the 2′-substituent (H, F, OMe). The 4′β-epimers are strongly destabilizing, but afford complete resistance against an exonuclease with the phosphate or phosphorothioate backbones. Crystal structures of RNA octamers containing 2′-F,4′-Cα-OMe-U, 2′-F,4′-Cβ-OMe-U, 2′-OMe,4′-Cα-OMe-U, 2′-OMe,4′-Cβ-OMe-U or 2′-F,4′-Cα-Me-U help rationalize these observations and point to steric and electrostatic origins of the unprecedented nuclease resistance seen with the chain-inverted 4′β-U epimer. We used structural models of human Argonaute 2 in complex with guide siRNA featuring 2′-F,4′-Cα-OMe-U or 2′-F,4′-Cβ-OMe-U at various sites in the seed region to interpret in vitro activities of siRNAs with the corresponding 2′-/4′-C-modifications.

Sequestering Ability of Oligophosphate Ligands toward Al3+ in Aqueous Solution

The interactions between Al3+ and ortho-phosphate (PO43–), pyro-phosphate (P2O74–, PP), tripolyphosphate (P3O105–, TPP) and hexa-metaphosphate (P6O186–, HMP) in aqueous solution have been studied. Formation constants, speciation models, and possible structures for the complexes formed are discussed on the basis of potentiometric, calorimetric, 31P–{1H} NMR, laser desorption mass spectrometric (LD MS) and MS/MS results. 31P–{1H} NMR experiments for Al3+–TPP system are in agreement with the speciation model drawn from analysis of potentiometric titration data, suggesting that TPP binds to Al3+ with two adjacent phosphate oxygen atoms, forming a six-membered ring. LD MS has provided identification and structural information for Al3+–PP, −TPP, and −HMP complexes which supports the reliability of speciation models proposed on the basis of the potentiometric data. Moreover, MS/MS spectra obtained from [ML]+ precursor ion show that the ion species involve linear HMP isomers and that two adjacent terminal phosphate groups probably bind Al3+. Standard enthalpy change values were obtained by titration calorimetry; all are endothermic, as is typical for hard–hard interactions. The dependence of formation constants on ionic strength over the range I = 0.1–1 mol kg–1 is also reported. The sequestering ability of all the ligands under study toward Al3+ was also evaluated by the empirical parameter pL0.5.

Increased intracellular Ca2+ concentrations prevent membrane localization of PH domains through the formation of Ca2+-phosphoinositides

Insulin resistance, a key etiological factor in metabolic syndrome, is closely linked to ectopic lipid accumulation and increased intracellular Ca2+ concentrations in muscle and liver. However, the mechanism by which dysregulated intracellular Ca2+ homeostasis causes insulin resistance remains elusive. Here, we show that increased intracellular Ca2+ acts as a negative regulator of insulin signaling. Chronic intracellular Ca2+ overload in hepatocytes during obesity and hyperlipidemia attenuates the phosphorylation of protein kinase B (Akt) and its key downstream signaling molecules by inhibiting membrane localization of pleckstrin homology (PH) domains. Pharmacological approaches showed that elevated intracellular Ca2+ inhibits insulin-stimulated Akt phosphorylation and abrogates membrane localization of various PH domain proteins such as phospholipase Cδ and insulin receptor substrate 1, suggesting a common mechanism inhibiting the membrane targeting of PH domains. PH domain-lipid overlay assays confirmed that Ca2+ abolishes the binding of various PH domains to phosphoinositides (PIPs) with two adjacent phosphate groups, such as PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 Finally, thermodynamic analysis of the binding interaction showed that Ca2+-mediated inhibition of targeting PH domains to the membrane resulted from the tight binding of Ca2+ rather than PH domains to PIPs forming Ca2+-PIPs. Thus, Ca2+-PIPs prevent the recognition of PIPs by PH domains, potentially due to electrostatic repulsion between positively charged side chains in PH domains and the Ca2+-PIPs. Our findings provide a mechanistic link between intracellular Ca2+ dysregulation and Akt inactivation in insulin resistance.

Structure-activity relationship study of ProxyPhos chemosensors for the detection of proximal phosphorylation and other phosphate species.

Chemosensors for the detection of phosphate-containing biological species are in high need. Detection of proximally phosphorylated sites of PPi and those found in peptides and proteins has been demonstrated using chemosensors containing pyrene, as a fluorescent reporter, and a Zn2+-chelate, as a phosphate-binding group. Using these sensors, detection of proximal phosphate groups is afforded by binding of at least two of the sensor molecules to the adjacent phosphates, via the Zn2+ centres, leading to excimer formation between the pyrene groups and the corresponding shift in emission from 376 to 476 nm. Although several reports of this chemosensor class have been made, no detailed studies of selectivity of these sensors among major phosphate targets have been reported. In this study, a library of this class of chemosensors, termed ProxyPhos, which contained various linkers and Zn2+-chelating groups (i.e. DPA, cyclen and cyclam), was prepared and the effects of structural variation on the sensing efficiency and selectivity were evaluated among proximally phosphorylated peptides, proteins, nucleotides, Pi and PPi. As a result of this study, we have identified ProxyPhos library members that are most suitable for the detection of proximally phosphorylated peptides, PPi, UTP, and a DpYD peptide motif, and have generally provided a foundation for the selection of ProxyPhos chemosensors for further development of specific biologically relevant assays. The broad utility of ProxyPhos is further supported by the demonstrated lack of these sensors' cytotoxicity, ability to rapidly permeate into live and fixed cells and compatibility with gel staining methods.

The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase.

The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase.

Insights into the activation mechanism of class I HDAC complexes by inositol phosphates.

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.

Identification of Toxoplasma TgPH1, a pleckstrin homology domain-containing protein that binds to the phosphoinositide PI(3,5)P2

The phosphoinositide phosphatidylinositol-3,5-bisphosphate (PI(3,5)P2) plays crucial roles in the maintenance of lysosome/vacuole morphology, membrane trafficking and regulation of endolysosome-localized membrane channel activity. In Toxoplasma gondii, we previously reported that PI(3,5)P2 is essential for parasite survival by controlling homeostasis of the apicoplast, a particular organelle of algal origin. Here, by using a phosphoinositide pull-down assay, we identified TgPH1 in Toxoplasma a protein conserved in many apicomplexan parasites. TgPH1 binds specifically to PI(3,5)P2, shows punctate intracellular localization, but plays no vital role for tachyzoite growth in vitro. TgPH1 is a protein predominantly formed by a pleckstrin homology (PH) domain. So far, PH domains have been described to bind preferentially to bis- or trisphosphate phosphoinositides containing two adjacent phosphates (i.e. PI(3,4)P2, PI(4,5)P2, PI(3,4,5)P3). Therefore, our study reveals an unusual feature of TgPH1 which binds preferentially to PI(3,5)P2.

Characterization of the dynamics in the protonic conductor CsH₂PO₄ by ¹⁷O solid-state NMR spectroscopy and first-principles calculations: correlating phosphate and protonic motion.

17O NMR spectroscopy combined with first-principles calculations was employed to understand the local structure and dynamics of the phosphate ions and protons in the paraelectric phase of the proton conductor CsH2PO4. For the room-temperature structure, the results confirm that one proton (H1) is localized in an asymmetric H-bond (between O1 donor and O2 acceptor oxygen atoms), whereas the H2 proton undergoes rapid exchange between two sites in a hydrogen bond with a symmetric double potential well at a rate ≥107 Hz. Variable-temperature 17O NMR spectra recorded from 22 to 214 °C were interpreted by considering different models for the rotation of the phosphate anions. At least two distinct rate constants for rotations about four pseudo C3 axes of the phosphate ion were required in order to achieve good agreement with the experimental data. An activation energy of 0.21 ± 0.06 eV was observed for rotation about the P–O1 axis, with a higher activation energy of 0.50 ± 0.07 eV being obtained for rotation about the P–O2, P–O3d, and P–O3a axes, with the superscripts denoting, respectively, dynamic donor and acceptor oxygen atoms of the H-bond. The higher activation energy of the second process is most likely associated with the cost of breaking an O1–H1 bond. The activation energy of this process is slightly lower than that obtained from the 1H exchange process (0.70 ± 0.07 eV) (Kim, G.; Blanc, F.; Hu, Y.-Y.; Grey, C. P. J. Phys. Chem. C2013, 117, 6504−6515) associated with the translational motion of the protons. The relationship between proton jumps and phosphate rotation was analyzed in detail by considering uncorrelated motion, motion of individual PO4 ions and the four connected/H-bonded protons, and concerted motions of adjacent phosphate units, mediated by proton hops. We conclude that, while phosphate rotations aid proton motion, not all phosphate rotations result in proton jumps.

Replacement of water molecules in a phosphate binding site by furanoside-appended lin-benzoguanine ligands of tRNA-guanine transglycosylase (TGT).

The enzyme tRNA-guanine transglycosylase has been identified as a drug target for the foodborne illness shigellosis. A key challenge in structure-based design for this enzyme is the filling of the polar ribose-34 pocket. Herein, we describe a novel series of ligands consisting of furanoside-appended lin-benzoguanines. They were designed to replace a conserved water cluster and differ by the functional groups at C(2) and C(3) of the furanosyl moiety being either OH or OMe. The unfavorable desolvation of Asp102 and Asp280, which are located close to the ribose-34 pocket, had a significant impact on binding affinity. While the enzyme has tRNA as its natural substrate, X-ray co-crystal structures revealed that the furanosyl moieties of the ligands are not accommodated in the tRNA ribose-34 site, but at the location of the adjacent phosphate group. A remarkable similarity of the position of the oxygen atoms in these two structures suggests furanosides as a potential phosphate isoster.

New crystal structures of HSC-70 ATP binding domain confirm the role of individual binding pockets and suggest a new method of inhibition.

In recent years the chaperone HSC-70 has become a target for drug design with a strong focus in anticancer therapies. In our study of possible inhibitors of HSC-70 enzymatic activity we screened compounds by NMR as well as X-ray crystallography. As part of our screening efforts we crystallized the human HSC-70 ATP binding domain and obtained novel crystal forms in addition to known structures. The new crystal structures highlight the mobility of the entire domain previously described by NMR, which was linked to its chaperone activity but not yet demonstrated by X-ray crystallography. Conformational changes across the entire molecule have been elucidated in response to the binding of small molecule ligands and show a pattern of mobility consistent with postulated signal transduction modes between the nucleotide binding domain (NBD) and the substrate binding domain (SBD). In addition, two crystal structures contained glycerol bound at a new site. Binding studies performed with glycerol analogs proved inhibitory properties of the site, which were further characterized by isothermal calorimetry and in silico docking studies. The presence of two binding pockets enabled us to explore a novel method of inhibition by compounds that bridge the adjacent phosphate and glycerol binding sites. Finally, an example of such a bridged inhibitor is proposed.

Amides are excellent mimics of phosphate internucleoside linkages and are well tolerated in short interfering RNAs.

RNA interference (RNAi) has become an important tool in functional genomics and has an intriguing therapeutic potential. However, the current design of short interfering RNAs (siRNAs) is not optimal for in vivo applications. Non-ionic phosphate backbone modifications may have the potential to improve the properties of siRNAs, but are little explored in RNAi technologies. Using X-ray crystallography and RNAi activity assays, the present study demonstrates that 3′-CH2-CO-NH-5′ amides are excellent replacements for phosphodiester internucleoside linkages in RNA. The crystal structure shows that amide-modified RNA forms a typical A-form duplex. The amide carbonyl group points into the major groove and assumes an orientation that is similar to the P–OP2 bond in the phosphate linkage. Amide linkages are well hydrated by tandem waters linking the carbonyl group and adjacent phosphate oxygens. Amides are tolerated at internal positions of both the guide and passenger strand of siRNAs and may increase the silencing activity when placed near the 5′-end of the passenger strand. As a result, an siRNA containing eight amide linkages is more active than the unmodified control. The results suggest that RNAi may tolerate even more extensive amide modification, which may be useful for optimization of siRNAs for in vivo applications.

The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase

Phosphoinositides are spatially restricted membrane signaling molecules. Phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2]--a phosphoinositide that is highly enriched in, and present throughout, the plasma membrane--has been implicated in endocytosis. Trypanosoma brucei has one of the highest known rates of endocytosis, a process it uses to evade the immune system. To determine whether phosphoinositides play a role in endocytosis in this organism, we have identified and characterized one of the enzymes that is responsible for generating PI(4,5)P2. Surprisingly, this phosphoinositide was found to be highly concentrated in the flagellar pocket, the only site of endocytosis and exocytosis in this organism. The enzyme (designated TbPIPKA, annotated as Tb927.10.1620) was present at the neck of the pocket, towards the anterior-end of the parasite. Depletion of TbPIPKA led to depletion of PI(4,5)P2 and enlargement of the pocket, the result of impaired endocytosis. Taken together, these data suggest that TbPIPKA and its product PI(4,5)P2 are important for endocytosis and, consequently, for homeostasis of the flagellar pocket.

The mechanism of human tyrosyl-DNA phosphodiesterase 1 in the cleavage of AP site and its synthetic analogs

The mechanism of hydrolysis of the apurinic/apyrimidinic (AP) site and its synthetic analogs by using tyrosyl-DNA phosphodiesterase 1 (Tdp1) was analyzed. Tdp1 catalyzes the cleavage of AP site and the synthetic analog of the AP site, 3-hydroxy-2(hydroxymethyl)-tetrahydrofuran (THF), in DNA by hydrolysis of the phosphodiester bond between the substituent and 5' adjacent phosphate. The product of Tdp1 cleavage in the case of the AP site is unstable and is hydrolyzed with the formation of 3'- and 5'-margin phosphates. The following repair demands the ordered action of polynucleotide kinase phosphorylase, with XRCC1, DNA polymerase β, and DNA ligase. In the case of THF, Tdp1 generates break with the 5'-THF and the 3'-phosphate termini. Tdp1 is also able to effectively cleave non-nucleotide insertions in DNA, decanediol and diethyleneglycol moieties by the same mechanism as in the case of THF cleavage. The efficiency of Tdp1 catalyzed hydrolysis of AP-site analog correlates with the DNA helix distortion induced by the substituent. The following repair of 5'-THF and other AP-site analogs can be processed by the long-patch base excision repair pathway.