The catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis

The Rev1-Polζ pathway is believed to be the major mechanism of translesion DNA synthesis and base damage-induced mutagenesis in eukaryotes. While it is widely believed that Rev1 plays a non-catalytic function in translesion synthesis, the role of its dCMP transferase activity remains uncertain. To determine the relevance of its catalytic function in translesion synthesis, we separated the Rev1 dCMP transferase activity from its non-catalytic function in yeast. This was achieved by mutating two conserved amino acid residues in the catalytic domain of Rev1, i.e. D467A/E468A, where its catalytic function was abolished but its non-catalytic function remained intact. In this mutant strain, whereas translesion synthesis and mutagenesis of UV radiation were fully functional, those of a site-specific 1,N⁶-ethenoadenine were severely deficient. Specifically, the predominant A→G mutations resulting from C insertion opposite the lesion were abolished. Therefore, translesion synthesis and mutagenesis of 1,N⁶-ethenoadenine require the catalytic function of the Rev1 dCMP transferase, in contrast to those of UV lesions, which only require the non-catalytic function of Rev1. These results show that the catalytic function of the Rev1 dCMP transferase is required in a lesion-specific manner for translesion synthesis and base damage-induced mutagenesis.     

Tautomerism and Regioselectivity in Ribosylation of Guanine

Abstract

N²-acetyl- and 9, N²-diacetylguanines were subjected to reaction with tetraacetylribose in the presence of p-toluenesulfonic acid. Unlike the ribosylation of diacetylguanine, which gives 7-riboside as a kinetic product, the reaction of monoacetylguanine produces directly a mixture of 7- and 9-ribosides. This reflects N⁷H ? N⁹H tautomerism of the guanine substrate and supports the hypothesis that only the unsubstituted nitrogens of the imidazolium portion of guanine (either N7 or N9) react directly with a sugar cation.     

AlkB Dioxygenase Preferentially Repairs Protonated Substrates: SPECIFICITY AGAINST EXOCYCLIC ADDUCTS AND MOLECULAR MECHANISM OF ACTION*

Efficient repair by Escherichia coli AlkB dioxygenase of exocyclic DNA adducts 3,N⁴-ethenocytosine, 1,N⁶-ethenoadenine, 3,N⁴-α-hydroxyethanocytosine, and reported here for the first time 3,N⁴-α-hydroxypropanocytosine requires higher Fe(II) concentration than the reference 3-methylcytosine. The pH optimum for the repair follows the order of pK_a values for protonation of the adduct, suggesting that positively charged substrates favorably interact with the negatively charged carboxylic group of Asp-135 side chain in the enzyme active center. This interaction is supported by molecular modeling, indicating that 1,N⁶-ethenoadenine and 3,N⁴-ethenocytosine are bound to AlkB more favorably in their protonated cationic forms. An analysis of the pattern of intermolecular interactions that stabilize the location of the ligand points to a role of Asp-135 in recognition of the adduct in its protonated form. Moreover, ab initio calculations also underline the role of substrate protonation in lowering the free energy barrier of the transition state of epoxidation of the etheno adducts studied. The observed time courses of repair of mixtures of stereoisomers of 3,N⁴-α-hydroxyethanocytosine or 3,N⁴-α-hydroxypropanocytosine are unequivocally two-exponential curves, indicating that the respective isomers are repaired by AlkB with different efficiencies. Molecular modeling of these adducts bound by AlkB allowed evaluation of the participation of their possible conformational states in the enzymatic reaction.     

Synthesis of 2-Chloro-6-aryloxy- and 2-Chloro-6-alkoxyarylpurines and Their Properties in the Purine Nucleoside Phosphorylase (PNP) System

Abstract

A series of 2-chloro-6-aryloxy- and 2-chloro-6-alkoxyarylpurines was synthesized and their kinetic properties in the purine nucleoside phosphorylase (PNP) system were determined. All compounds showed inhibitory activity (IC₅₀ in the range 0.5-76 μM) vs. hexameric (“high-molecular weight”) PNP from E. coli. By contrast, no inhibition vs. trimeric Cellulomonas PNP was detected.     

Synthesis of 6-aryloxy- and 6-arylalkoxy-2-chloropurines and their interactions with purine nucleoside phosphorylase from Escherichia coli

The phase transfer method was applied to perform the nucleophilic substitution of 2,6-dichloropurines by modified arylalkyl alcohol or phenols. Since under these conditions only the 6-halogen is exchanged, this method gives 2-chloro-6-aryloxy- and 2-chloro-6-arylalkoxy-purines. 2-Chloro-6-benzylthiopurine was synthesized by alkylation of 2-chloro-6-thiopurine with benzyl bromide. The stereoisomers of 2-chloro-6-(1-phenyl-1-ethoxy)purine were obtained from R- and S-enantiomers of sec.-phenylethylalcohol and 2,6-dichloropurine. All derivatives were tested for inhibition with purified hexameric E. coli purine nucleoside phosphorylase (PNP). For analogues showing IC50 < 10 microM, the type of inhibition and inhibition constants were determined. In all cases the experimental data were best described by the mixed-type inhibition model and the uncompetitive inhibition constant, Kiu, was found to be several-fold lower than the competitive inhibition constant, Kic. This effect seems to be due to the 6-aryloxy- or 6-arylalkoxy substituent, because a natural PNP substrate adenine, as well as 2-chloroadenine, show mixed type inhibition with almost the same inhibition constants Kiu and Kic. The most potent inhibition was observed for 6-benzylthio-2-chloro-, 6-benzyloxy-2-chloro-, 2-chloro-6-(2-phenyl-1-ethoxy), 2-chloro-6-(3-phenyl-1-propoxy)- and 2-chloro-6-ethoxypurines (Kiu = 0.4, 0.6, 1.4, 1.4 and 2.2 microM, respectively). The R-stereoisomer of 2-chloro-6-(1-pheny-1-ethoxy)purine has Kiu = 2.0 microM, whereas inhibition of its S counterpart is rather weak (IC50 > 12 microM). More rigid (e.g. phenoxy-), non-planar (cyclohexyloxy-), or more bulky (2,4,6-trimethylphenoxy-) substituents at position 6 of the purine base gave less potent inhibitors (IC50 = 26, 56 and > 100 microM, respectively). The derivatives are selective inhibitors of hexameric "high-molecular mass" PNPs because no inhibitory activity vs. trimeric Cellulomonas sp. PNP was detected. By establishing the ligand-dependent stabilization pattern of the E. coli PNP it was shown that the new derivatives, similarly as the natural purine bases, are able to form a dead-end ternary complex with the enzyme and orthophosphate. It was also shown that the derivatives are substrates in the reverse synthetic direction catalyzed by E. coli PNP.     

Synthesis of a novel pyridine-diamino bridged diphosphine ligand and its macrocyclic metal complexes

A novel long chain diphosphine ligand with a pyridine-diamino bridge, 2,6-bis(N-benzyl-N-diphenylphosphinomethylamino)pyridine (PNP1), was prepared conveniently using the Mannich reaction of HPPh₂ with paraformaldehyde and 2,6-bis(N-benzylamino)pyridine in high yield. Reactions of the ligand with metal complexes, M(COD)Cl₂ (M = Pd, Pt), M(CH₃CN)₄ClO₄ (M = Cu, Ag) and M(CO)₆ (M = Mo, W) afforded the corresponding 10-numbered monometallic macrocyclic complexes with an uncoordinated pyridyl bridge. The monometallic chelate PdCl₂(PNP1) continued to react with Ag⁺ or Cu⁺ giving the μ-Cl bridged bicyclic metallic complex (μ-Cl)₂[PdCl(PNP1)]₂. The diphenylphosphine group coordinated with metal ion in cis-form in all the 10-numbered macrocyclic metal complexes. Ligand PNP1 and another known analogous 2,6-bis(N-diphenylphosphinoamino)pyridine (PNP2) reacted with Au(SMe₂)Cl giving the corresponding bimetallic Au₂Cl₂(PNP1) and Au₂Cl₂(PNP2), respectively. The latter bimetallic complexes continued to react with Ag⁺ and diphosphine ligand to give the corresponding bimetallic macrocyclic complexes Au₂(ligand)₂(ClO₄)₂. All the complexes were characterized and the structures of some complexes were confirmed by X-ray single crystallography determination.     

Formycins A and B and some analogues: selective inhibitors of bacterial (Escherichia coli) purine nucleoside phosphorylase.

Formycin B (FB), a moderate inhibitor (Ki approximately 100 microM) of mammalian purine nucleoside phosphorylase (PNP), and formycin A (FA), which is totally inactive vs. the mammalian enzyme, are both effective inhibitors of the bacterial (Escherichia coli) enzyme (Ki approximately 5 microM). Examination of a series of N-methyl analogues of FA and FB led to the finding that N(6)-methyl-FA, virtually inactive vs. the mammalian enzyme, is the most potent inhibitor of E. coli purine nucleoside phosphorylase (Ki approximately 0.3 uM) at neutral pH. Inhibition is competitive not only with respect to Ino, but also relative to 7-methyl-Guo and 7-methyl-Ado, as substrates. Both oxoformycins A and B are relatively poor inhibitors. For the most potent inhibitor, N(6)-methyl-FA, it was shown that the enzyme preferentially binds the neutral, and not the cationic, form. In accordance with this the neutral, but not the cationic form, of the structurally related N(1)-methyl-Ado was found to be an excellent substrate. Reported data on tautomerism of formycins were profited from, and extended, to infer which tautomeric species and ionic forms are the active inhibitors. A commercially available (Sigma) bacterial PNP, of unknown origin, was shown to differ from the E. coli enzyme by its inability to phosphorylase Ado; this enzyme was also resistant to FA and FB. These findings have been extended to provide a detailed comparison of the substrate/inhibitor properties of PNP from various microorganisms.     

Fluorescent analogs of NAADP with calcium mobilizing activity

Nicotinic acid adenine dinucleotide phosphate (NAADP) mobilizes Ca²⁺ through a mechanism totally independent of cyclic ADP-ribose or inositol trisphosphate. Fluorescent analogs of NAADP were synthesized in this study to facilitate further characterization of this novel Ca²⁺ release mechanism. The base-exchange reaction catalyzed by ADP-ribosyl cyclase was utilized to convert nicotinamide 1,N⁶-ethenoadenine dinucleotide phosphate to a fluorescent product, nicotinic acid 1,N⁶-ethenoadenine dinucleotide phosphate (etheno-NAADP). The excitation spectrum of the product showed two maxima at 275 nm and 300 nm and an emission maximum at 410 nm. An aza derivative of etheno-NAADP was also synthesized by sequential treatments with NaOH and nitrite. The product, nicotinic acid 1,N⁶-etheno-2-aza-adenine dinucleotide phosphate (etheno-aza-NAADP) had excitation maxima at 280 nm and 360 nm and an emission maximum at 470 nm. The fluorescence of both analogs was sensitive to polarity and exhibited a 3–4-fold enhancement going from an aqueous buffer to an organic solvent. Proton-NMR measurements confirmed the presence of the etheno ring in both analogs. In the aza derivative the proton at the 2-position of the adenine ring was absent, consistent with the conversion of the 2-carbon to a nitrogen. Both analogs could activate Ca²⁺ release from sea urchin egg homogenates and the half-maximal concentrations for etheno-aza-NAADP and etheno-NAADP were at about 2.5 μM and 5 μM, respectively. At sub-threshold concentrations, both analogs could also function as antagonists, inactivating the NAADP-sensitive Ca²⁺ release with a half-maximal concentration of 60–80 nM. Microinjection of etheno-aza-NAADP into live eggs activated Ca²⁺ increase and triggered a cortical exocytotic reaction confirming its effectiveness in vivo. These fluorescent analogs are potentially useful for visualizing the novel Ca²⁺ stores that are sensitive to NAADP in live cells.     

New aromatic 6-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives as potential plant growth regulators

Cytokinins are naturally occurring substances that act as plant growth regulators promoting plant growth and development, including shoot initiation and branching, and also affecting apical dominance and leaf senescence. Aromatic cytokinin 6-benzylaminopurine (BAP) has been widely used in micropropagation systems and biotechnology. However, its 9-glucoside (BAP9G) accumulates in explants, causing root inhibition and growth heterogenity. To overcome BAP disadvantages, a series of ring-substituted 2′-deoxy-9-(β)-d-ribofuranosylpurine derivatives was prepared and examined in different classical cytokinin bioassays. Amaranthus, senescence and tobacco callus bioassays were employed to provide details of cytokinin activity of 2′-deoxy-9-(β)-d-ribosides compared to their respective free bases and ribosides. The prepared derivatives were also tested for their recognition by cytokinin receptors of Arabidopsis thaliana AHK3 and CRE1/AHK4. The ability of aromatic N⁶-substituted adenine-2′-deoxy-9-(β)-d-ribosides to promote plant growth and delay senescence was increased considerably and, in contrast to BAP, no loss of cytokinin activity at higher concentrations was observed. The presence of a 2′-deoxyribosyl moiety at the N9-position led to an increase in cytokinin activities in comparison to the free bases and ribosides. The antioxidant capacity, cytotoxicity and effect on the MHV-68 gammaherpesvirus strain were also examined.     

The pyrimidine ring-opened derivative of 1,N6-ethenoadenine is excised from DNA by the Escherichia coli Fpg and Nth proteins

It was previously shown that 1,N(6)-ethenoadenine (epsilonA) in DNA rearranges into a pyrimidine ring-opened derivative of 20-fold higher mutagenic potency in Escherichia coli (AB1157 lacDeltaU169) than the parental epsilonA (Basu, A. K., Wood, M. L., Niedernhofer, L. J., Ramos, L. A., and Essigmann, J. M. (1993) Biochemistry 32, 12793-12801). We have found that at pH 7.0, the stability of the N-glycosidic bond in epsilondA is 20-fold lower than in dA. In alkaline conditions, but also at neutrality, epsilondA depurinates or converts into products: epsilondA --> B --> C --> D. Compound B is a product of water molecule addition to the C(2)-N(3) bond, which is in equilibrium with a product of N(1)-C(2) bond rupture in epsilondA. Compound C is a deformylated derivative of ring-opened compound B, which further depurinates yielding compound D. Ethenoadenine degradation products are not recognized by human N-alkylpurine-DNA glycosylase, which repairs epsilonA. Product B is excised from oligodeoxynucleotides by E. coli formamidopyrimidine-DNA glycosylase (Fpg) and endonuclease III (Nth). Repair by the Fpg protein is as efficient as that of 7,8-dihydro-8-oxoguanine when the excised base is paired with dT and dC but is less favorable when paired with dG and dA. Ethenoadenine rearrangement products are formed in oligodeoxynucleotides also at neutral pH at the rate of about 2-3% per week at 37 degrees C, and therefore they may contribute to epsilonA mutations.     

Mass-spectrometric quantification of cytokinin nucleotides and glycosides in tobacco crown-gall tissue

the cytokinins of tobacco crown-gall tissue have been analysed by quantitative mass spectrometry using ²H₂-labelled cytokinin riboside 5-monophosphates and ¹⁵N₄-labelled cytokinin glycosides as internal standards. The principal endogenous cytokinin of this tissue is zeatin riboside 5-monophosphate. The biologically inactive 7-glucoside of zeatin is the most abundant basic cytokinin in the tissue. These findings expose the limitations of previously reported analyses of similar tissues, which were restricted to biologically active basic cytokinins. The present study demonstrates that the endogenous cytokinins of tobacco crowngall tissue show a clear correspondence to the range of metabolites formed when exogenous cytokinins are supplied to nontumorous tobacco cells.Abbreviations DHZ dihydrozeatin - DHZ7G dihydrozeatin 7-glucoside - DHZMP dihydrozeatin 9-riboside 5-monophosphate - DHZR dihydrozeatin 9-riboside - GC-MS coupled gas chromatography-mass spectrometry - HPLC high-performance liquid chromatography - Z7G zeatin 7-glucoside - Z9G zeatin 9-glucoside - ZOG zeatin O-glucoside - ZMP zeatin 9-riboside 5-monophosphate - ZR zeatin 9-riboside - ZROG zeatin 9-riboside O-glucoside     

AAA+ ATPase p97/VCP mutants and inhibitor binding disrupt inter-domain coupling and subsequent allosteric activation

The human AAA+ ATPase p97, also known as valosin-containing protein, a potential target for cancer therapeutics, plays a vital role in the clearing of misfolded proteins. p97 dysfunction is also known to play a crucial role in several neurodegenerative disorders, such as MultiSystem Proteinopathy 1 (MSP-1) and Familial Amyotrophic Lateral Sclerosis (ALS). However, the structural basis of its role in such diseases remains elusive. Here, we present cryo-EM structural analyses of four disease mutants p97^R155H, p97^R191Q, p97^A232E, p97^D592N, as well as p97^E470D, implicated in resistance to the drug CB-5083, a potent p97 inhibitor. Our cryo-EM structures demonstrate that these mutations affect nucleotide-driven allosteric activation across the three principal p97 domains (N, D1, and D2) by predominantly interfering with either (1) the coupling between the D1 and N-terminal domains (p97^R155H and p97^R191Q), (2) the interprotomer interactions (p97^A232E), or (3) the coupling between D1 and D2 nucleotide domains (p97^D592N, p97^E470D). We also show that binding of the competitive inhibitor, CB-5083, to the D2 domain prevents conformational changes similar to those seen for mutations that affect coupling between the D1 and D2 domains. Our studies enable tracing of the path of allosteric activation across p97 and establish a common mechanistic link between active site inhibition and defects in allosteric activation by disease-causing mutations and have potential implications for the design of novel allosteric compounds that can modulate p97 function.     

Sensitivity of human type II topoisomerases to DNA damage: stimulation of enzyme-mediated DNA cleavage by abasic, oxidized and alkylated lesions

Type II topoisomerases are essential enzymes that are also the primary cellular targets for a number of important anticancer drugs. These drugs act by increasing levels of topoisomerase II-mediated DNA cleavage. Recent studies indicate that endogenous forms of DNA damage, such as abasic sites and base mismatches, also stimulate the DNA scission activity of the enzyme. To extend our understanding of how type II topoisomerases react to DNA damage, the effects of abasic sites, and oxidized and alkylated bases on DNA cleavage mediated by human topo-isomerase IIα and β were determined. Based on experiments that incorporated random abasic sites into plasmid DNA, human type II enzymes can locate lesions even within a background of several thousand undamaged base pairs. As determined by experiments that utilized site-specific forms of DNA lesions, oxidized or monoalkylated purines that allow base pairing and induce little distortion in the double helix have modest effects on topoisomerase II-mediated DNA cleavage. In contrast, 1,N⁶-ethenoadenine, a bulky lesion that disrupts base pairing, enhanced DNA cleavage ~10-fold. 1,N⁶-Ethenoadenine is the first lesion found to rival the stimulatory effects of apurinic sites on the DNA scission activity of eukaryotic type II topoisomerases.     

通过染色体整合表达古菌乙酰化酶合成N-末端乙酰化胸腺肽β4

胸腺肽β4（Tβ4）是N-末端乙酰化的43肽,具有多种重要生物学功能.其生物合成存在两大难点,即乙酰化修饰和小分子肽的表达.本研究发现来自古菌Sulfolobus solfataricus的乙酰化酶ssArd1可以催化Tβ4的N-末端乙酰化修饰.利用Red同源重组技术将ssArd1基因表达盒整合至E.coli BL21（DE3）染色体的lpxM位点上,构建了可以实现Tβ4N-末端乙酰化修饰的新型宿主E.coli BDA.将Tβ4编码基因融合在改造的微型Spl DnaX Intein的N端,并在Intein的C端添加His标签,构建了表达载体pET-Tβ4-Intein.在E.coli BDA中表达的融合蛋白,经镍亲和层析纯化后用β-巯基乙醇诱导融合蛋白切割释放小分子多肽,获得了具有N-末端乙酰化的Tβ4.     

In vitro cytokinin binding to a particulate cell fraction from protonemata of Funaria hygrometrica

Cytokinin-induced bud formation in moss protonemata is specific for cytokinin bases, their ribosides being relatively inactive. Binding of [³H]benzyladenine (BA) to a 13,000–80,000 x g subcellular fraction from extracts of Funaria hygrometrica (L.) Sibth. was measured by a centrifugation assay. Increasing concentrations of non-radioactive BA decreased the binding proportionally to the logarithm of the BA concentration between 3×10^-8 and 10^-4M. [³H]Zeatin also bound to these fractions, although the extent of binding was not as great as with [³H]BA. Biologically active cytokinins, including BA, zeatin, 6-(3-methyl-2-enylamino)purine (IPA) and kinetin, competed for the binding of [³H]BA, whereas the ribosides of BA, zeatin and IPA competed poorly. Other biologically inactive compounds, such as adenine and 9-methyl-BA, were also ineffective as competitors. The ability to bind BA by the 13,000–80,000 x g fraction was greatly reduced by treatment with 1% Triton X-100, and heat treatment eliminated more than one-half of the binding activity. Competitive binding appeared to be pH-dependent, with maximal activity between pH 6.0 and 6.5. After fractionation by differential centrifugation, the ability to bind cytokinins was not correlated with the RNA content of the fraction and thus probably did not represent binding to ribosomes which has been reported in other plant tissues. Cytokinins also exhibited competitive binding to non-biological materials, e.g., talc. The detailed characteristics of the binding of BA to talc were different from those to the biological fractions. However, the problem remains, in all studies of cytokinin binding, to distinguish between binding that is biologically meaningful, and biological (biologically) non-meaningful physical adsorption.Abbreviations BA N⁶-benzyladenine - IPA 6-(3-methyl-2-enylamino)purine - 9-MeBA N⁶-benzyl-9-methyladenine     

Cytokinins: A Rapid Extraction and Purification Method

A method is described for the isolation, purification and quantitation of free cytokinin bases and ribosides using ethyl acetate at pH 7.7 for the extraction. The extraction is almost complete (97.7%) as determined by using N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C. The subsequent fast purification by chromatography on a standardized silica gel column in chloroform-methanol (7:3 v/v) is followed by thin layer chromatography (silica gel 60 F²⁵⁴) in chloroform-acetic acid (8:2 v/v). The recovery of N⁶-(Δ²-isopentenyl)adenine-8-¹⁴C after this two step purification was 78–82%. The efficiency of the method was determined by applying this procedure to N⁶-(Δ²-isopentenyl)adenine and N⁶(Δ²-isopentenyl)adenosine. Using gas liquid chromatography the recovery for N⁶-(Δ²-isopentenyl)adenosine was determined to be 61% and compared to 43% for N⁶-(Δ²-isopentenyl)adenosine, showing the suitability of the described method for gas liquid chromatography.     

Induced synthesis of formyltetrahydrofolate synthetase in Micrococcus aerogenes

Summary The levels of formyltetrahydrofolate synthetase and cyclohydrolase in M. aerogenes were enhanced 3-to 10-fold by growth in media containing formate of histidine. This induced synthesis was decreased by the simultaneous addition of ribosides or ribotides. Histidine, but not formate, also induced the synthesis of formimino transferase and/or cyclodeaminase. The specific activities of N¹⁰-formyltetrahydrofolate deacylase, serine hydroxymethylase and N⁵, N¹⁰-methylenetetrahydrofolate dehydrogenase were not affected by formate or histidine. These observations have been discussed with respect to the known mechanisms of regulation of tetrahydrofolate linked enzymes.Dedicated to Prof. C. B. van Niel on the occasion of his 70th birthday.Recipient of Research Career Award GM-K6-422.     

Response of human REV1 to different DNA damage: preferential dCMP insertion opposite the lesion

REV1 functions in the DNA polymerase ζ mutagenesis pathway. To help understand the role of REV1 in lesion bypass, we have examined activities of purified human REV1 opposite various template bases and several different DNA lesions. Lacking a 3′→5′ proofreading exonuclease activity, purified human REV1 exhibited a DNA polymerase activity on a repeating template G sequence, but catalyzed nucleotide insertion with 6-fold lower efficiency opposite a template A and 19–27-fold lower efficiency opposite a template T or C. Furthermore, dCMP insertion was greatly preferred regardless of the specific template base. Human REV1 inserted a dCMP efficiently opposite a template 8-oxoguanine, (+)-trans-anti-benzo[a]pyrene-N ²-dG, (–)-trans-anti-benzo[a]pyrene-N ²-dG and 1,N ⁶-ethenoadenine adducts, very inefficiently opposite an acetylaminofluorene-adducted guanine, but was unresponsive to a template TT dimer or TT (6–4) photoproduct. Surprisingly, the REV1 specificity of nucleotide insertion was very similar in response to different DNA lesions with greatly preferred C insertion and least frequent A insertion. By combining the dCMP insertion activity of human REV1 with the extension synthesis activity of human polymerase κ, bypass of the trans-anti-benzo[a]pyrene-N ² -dG adducts and the 1,N ⁶-ethenoadenine lesion was achieved by the two-polymerase two-step mechanism. These results suggest that human REV1 is a specialized DNA polymerase that may contribute to dCMP insertion opposite many types of DNA damage during lesion bypass.     

Structural basis for the function of pyridoxine 5'-phosphate synthase

BACKGROUND: Pyridoxal 5'-phosphate is the active form of vitamin B(6) that acts as an essential, ubiquitous coenzyme in amino acid metabolism. In Escherichia coli, the pathway of the de novo biosynthesis of vitamin B(6) results in the formation of pyridoxine 5'-phosphate (PNP), which can be regarded as the first synthesized B(6) vitamer. PNP synthase (commonly referred to as PdxJ) is a homooctameric enzyme that catalyzes the final step in this pathway, a complex intramolecular condensation reaction between 1-deoxy-D-xylulose-5'-phosphate and 1-amino-acetone-3-phosphate. RESULTS: The crystal structure of E. coli PNP synthase was solved by single isomorphous replacement with anomalous scattering and refined at a resolution of 2.0 A. The monomer of PNP synthase consists of one compact domain that adopts the abundant TIM barrel fold. Intersubunit contacts are mediated by three additional helices, respective to the classical TIM barrel helices, generating a tetramer of symmetric dimers with 422 symmetry. In the shared active sites of the active dimers, Arg20 is directly involved in substrate binding of the partner monomer. Furthermore, the structure of PNP synthase with its physiological products, PNP and P(i), was determined at 2.3 A resolution, which provides insight into the dynamic action of the enzyme and allows us to identify amino acids critical for enzymatic function. CONCLUSION: The high-resolution structures of the free enzyme and the enzyme-product complex of E. coli PNP synthase suggest essentials of the enzymatic mechanism. The main catalytic features are active site closure upon substrate binding by rearrangement of one C-terminal loop of the TIM barrel, charge-charge stabilization of the protonated Schiff-base intermediate, the presence of two phosphate binding sites, and a water channel that penetrates the beta barrel and allows the release of water molecules in the closed state. All related PNP synthases are predicted to fold into a similar TIM barrel pattern and have comparable active site architecture. Thus, a common mechanism can be anticipated.     

Using genotype × nitrogen interaction variables to evaluate the QTL involved in wheat tolerance to nitrogen constraints

Lower market prices and environmental concerns now orientate wheat (Triticum aestivum L.) breeding programs towards low input agricultural practices, and more particularly low nitrogen (N) input management. Such programs require knowledge of the genetic determination of plant reaction to N deficiency. Our aim was to characterize the genetic basis of N use efficiency and genotype × N interactions. The detection of QTL for grain yield, grain protein yield and their components was performed on a mapping population of 222 doubled haploid lines (DH), obtained from the cross between an N stress tolerant variety and an N stress sensitive variety. Experiments on the population were carried out in seven different environments, and in each case under high (N⁺) and low (N⁻) N supplies. In total, 233 QTL were detected for traits measured in each combination of environment and N supply, for “global” interaction variables (N⁺–N⁻ and N⁻/N⁺), for sensitivity to N stress and for performance under N-limited conditions which were assessed using factorial regression parameters. The 233 QTL were detected on the whole genome and clustered into 82 genome regions. The dwarfing gene (Rht-B1), the photoperiod sensitivity gene (Ppd-D1) and the awns inhibitor gene (B1) coincided with regions that contained the highest numbers of QTL. Non-interactive QTL were detected on linkage groups 3D, 4B, 5A1 and 7B2. Interactive QTL were revealed by interaction or factorial regression variables (2D2, 3D, 5A1, 5D, 6A, 6B, 7B2) or by both variables (1B, 2A1, 2A2, 2D1, 4B, 5A2, 5B). The usefulness of QTL meta-analysis and factorial regression to study QTL × N interactions and the impact of Rht-B1, Ppd-D1 and B1, are discussed. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.