Conferring a template-dependent polymerase activity to terminal deoxynucleotidyltransferase by mutations in the Loop1 region

Terminal deoxynucleotidyltransferase (Tdt) and DNA polymerase μ (pol μ) are two eukaryotic highly similar proteins involved in DNA processing and repair. Despite their high sequence identity, they differ widely in their activity: pol μ has a templated polymerase activity, whereas Tdt has a non-templated one. Loop1, first described when the Tdt structure was solved, has been invoked as the major structural determinant of this difference. Here we describe attempts to transform Tdt into pol μ with the minimal number of mutations in and around Loop1. First we describe the effect of mutations on six different positions chosen to destabilize Tdt Loop1 structure, either by alanine substitution or by deletion; they result at most in a reduction of Tdt activity, but adding Co⁺⁺ restores most of this Tdt activity. However, a deletion of the entire Loop1 as in pol λ does confer a limited template-dependent polymerase behavior to Tdt while a chimera bearing an extended pol μ Loop1 reproduces pol μ behavior. Finally, 16 additional substitutions are reported, targeted at the two so-called ‘sequence determinant’ regions located just after Loop1 or underneath. Among them, the single-point mutant F401A displays a sequence-specific replicative polymerase phenotype that is stable upon Co⁺⁺ addition. These results are discussed in light of the available crystal structures.     

The influence of internal and skin temperatures on active cutaneous vasodilation under different levels of exercise and ambient temperatures in humans

To clarify the influence of internal and skin temperature on the active cutaneous vasodilation during exercise, the body temperature thresholds for the onset of active vasodilation during light or moderate exercise under different ambient temperature conditions were compared. Seven male subjects performed 30 min of a cycling exercise at 20 % or 50 % of peak oxygen uptake in a room maintained at 20, 24, or 28 °C. Esophageal (Tes) and mean skin temperature (Tsk) as measured by a thermocouple, deep thigh temperature (Tdt) by the zero-heat-flow (ZHF) method, and forearm skin blood flow by laser-Doppler flowmetry (LDF) were monitored. The mean arterial pressure (MAP) was also monitored non-invasively, and the cutaneous vascular conductance (CVC) was calculated as the LDF/MAP. Throughout the experiment, the Tsk at ambient temperatures of 20, 24, and 28 °C were approximately 30, 32, and 34 °C, respectively, for both 20 % and 50 % exercise. During 50 % exercise, the Tes or Tdt thresholds for the onset of the increase in CVC were observed to be similar among the 20, 24, and 28 °C ambient conditions. During 20 % exercise, the increase in Tes and Tdt was significantly lower than those found at 50 %, and the onset of the increase in CVC was only observed at 28 °C. These results suggest that the onset of active vasodilation was affected more strongly by the internal or exercising tissue temperatures than by the skin temperatures during exercise performed at a moderate load in comparison to a light load under Tsk variations ranging from 30 °C to 34 °C. Therefore, the modification by skin temperature of the central control on cutaneous vasomotor tone during exercise may differ between different exercise loads.     

Structural basis for a novel mechanism of DNA bridging and alignment in eukaryotic DSB DNA repair

Eukaryotic DNA polymerase mu of the PolX family can promote the association of the two 3′‐protruding ends of a DNA double‐strand break (DSB) being repaired (DNA synapsis) even in the absence of the core non‐homologous end‐joining (NHEJ) machinery. Here, we show that terminal deoxynucleotidyltransferase (TdT), a closely related PolX involved in V(D)J recombination, has the same property. We solved its crystal structure with an annealed DNA synapsis containing one micro‐homology (MH) base pair and one nascent base pair. This structure reveals how the N‐terminal domain and Loop 1 of Tdt cooperate for bridging the two DNA ends, providing a templating base in trans and limiting the MH search region to only two base pairs. A network of ordered water molecules is proposed to assist the incorporation of any nucleotide independently of the in trans templating base. These data are consistent with a recent model that explains the statistics of sequences synthesized in vivo by Tdt based solely on this dinucleotide step. Site‐directed mutagenesis and functional tests suggest that this structural model is also valid for Pol mu during NHEJ.     

Relationship between mean body temperature calculated by two- or three-compartment models and active cutaneous vasodilation in humans: a comparison between cool and warm environments during leg exercise

The aim of this study was to assess whether the three-compartment model of mean body temperature (Tb3) calculated from the esophageal temperature (Tes), temperature in deep tissue of exercising muscle (Tdt), and mean skin temperature (Tsk) has the potential to provide a better match with the thermoregulatory responses than the two-component model of mean body temperature (Tb2) calculated from Tes and Tsk. Seven male subjects performed 40 min of a prolonged cycling exercise at 30% maximal oxygen uptake at 21°C or 31°C (50% relative humidity). Throughout the experiment, Tsk, Tb2, Tb3, and Tdt were significantly (P < 0.01) lower at 21°C than at 31°C temperature conditions, while Tes was similar under both conditions. During exercise, an increase in cutaneous vascular conductance (skin blood flow / mean arterial pressure) over the chest (%CVCc) was observed at both 21°C and 31°C, while no increase was observed at the forearm at 21°C. Furthermore, the Tb3 and Tdt threshold for the onset of the increase in %CVCc was similar, but the Tes and Tb2 threshold differed significantly (P < 0.05) between the conditions tested. These results suggest that active cutaneous vasodilation at the chest is related more closely to Tb3 or Tdt than that measured by Tes or Tb2 calculated by Tes and Tsk during exercise at both 21°C and 31°C.     

A comprehensive immunotopographic map of human thymus

We have achieved a comprehensive immunotopographic mapping of human thymus by using a large battery of monoclonal antibodies and the methodological refinement of comparative serial tissue section immunohistochemistry, allowing analysis of multiple phenotypes in the same tissue site. Previous immunohistochemical studies of thymus have concentrated on the majority T-cell and epithelial cell populations. Besides demonstrating the complexity of T-cell antigenic expression (e.g., simultaneous cortical expression of Leu 2, Leu 3, CALLA, Tdt, and Leu 6), we delineate surprisingly complex B-cell zones (e.g., septal B-follicles with DRC+C3d+ dendritic cells and zonal maturation of B-cells). Whereas septal B-follicles were found in 25% of cases, medullary B-cells were universally present as a substantial minority component. This expanded immunotopographic knowledge of the complex T-, B-, epithelial, and reticulum cell neighborhoods suggests that the thymus is an organ capable of a broad repertoire of immunological responses, not limited to T-cell development.     

Maternal behavior regulates long-term hippocampal expression of BAX and apoptosis in the offspring

Naturally occurring variations in maternal care influence hippocampal development in the rat. In the present study we found that variations in maternal licking/grooming (LG) during the first week of life are associated with altered hippocampal expression of BAX (group-1 tumor necrosis factor family mediated cell death effector) in 90-day-old male offspring. BAX-like immunoreactivity on western blots is significantly increased in the adult offspring of low-level LG mothers. There is no effect of maternal care on levels of either B-cell lymphoma-2 (BCL-2) (group-II mitochondria mediated cell death suppressor) or BAD (group-III endoplasmic reticulum mediated cell death effector). The most striking biochemical event in apoptosis is DNA fragmentation. Terminal deoxynucleotidyl transerferase (Tdt)-mediated dUTP-biotin nick-end labeling (TUNEL) and 4',6'-diamidino-2-phenylindole hydrochloride (DAPI) staining showed that the number of TUNEL-positive cells in both the dentate gyrus and CA1 region of the hippocampus is significantly increased in the adult offspring of low-level LG mothers. In conclusion, we propose that hippocampal neurons in the offspring of low-level LG mothers may be more vulnerable to loss through apoptosis.     

Unmasking of an essential thiol during function of the membrane-bound enzyme II of the phosphenolpyruvate beta-glucoside phosphotransferase system of Escherichia coli.

beta-Glucoside transport by phosphoenolpyruvate-hexose phosphotransferase system in Escherichia coli is inactivated in vivo by thiol reagents. This inactivation is strongly enhanced by the presence of transported substrates. In a system reconstituted from soluble and membrane-bound components, only the particulate component, the membrane-bound enzyme IIbgl appeared as the target of N-ethylmaleimide inaction. The same feature was found in the case of methyl-alpha-D-glucoside uptake via enzyme IIglc. It is shown that the sensitizing effect of substrates is specific and not generalized, methyl-alpha-D-glucoside only sensitizes enzyme IIglc and p-nitrophenyl-beta-D-glucoside only sensitizes enzyme IIbgl towards N-ethylmaleimide inactivation. The inactivation of enzyme IIbgl by thiol reagents is also promoted in vivo by fluoride inhibition of phosphoenolpyruvate synthesis. In toluene-treated bacteria, the presence of phosphoenolpyruvate protects against inactivation by thiol reagents of p-nitrophenyl-beta-D-glucoside phosphorylation. Both results suggest that the inactivator resistent form of enzyme IIbgl is an energized form of the enzyme.     

Diffusion control in reversible enzyme reactions. Applications to carbonic anhydrase.

The limit to the possible rate of reversible enzymatic reactions set by the diffusional motion has been considered. It is found that not only the diffusion of the reactants to the enzyme but also the diffusion away of the products can be rate limiting. To avoid assumptions about the detailed nature of the enzyme only diffusion in the bulk aqueous medium is treated. By such an approach one obtains an upper limit to the possible rate. In the latter half of the paper the derived general equations are applied to the possible suggested reaction schemes for the enzyme carbonic anhydrase. It is found that a scheme involving HCO3- as substrate for the dehydration process and a direct reaction between buffer and enzyme is comsistent with the limits set by the diffusional motion, while several other possibilities can be ruled out.     

Structures of Intermediates along the Catalytic Cycle of Terminal Deoxynucleotidyltransferase: Dynamical Aspects of the Two-Metal Ion Mechanism

Terminal deoxynucleotidyltransferase (Tdt) is a non-templated eukaryotic DNA polymerase of the polX family that is responsible for the random addition of nucleotides at the V(D)J junctions of immunoglobulins and T-cell receptors. Here we describe a series of high-resolution X-ray structures that mimic the pre-catalytic state, the post-catalytic state and a competent state that can be transformed into the two other ones in crystallo via the addition of dAMPcPP and Zn^2 +, respectively. We examined the effect of Mn^2 +, Co^2 + and Zn^2 + because they all have a marked influence on the kinetics of the reaction. We demonstrate a dynamic role of divalent transition metal ions bound to site A: (i) Zn^2 + (or Co^2 +) in Metal A site changes coordination from octahedral to tetrahedral after the chemical step, which explains the known higher affinity of Tdt for the primer strand when these ions are present, and (ii) metal A has to leave to allow the translocation of the primer strand and to clear the active site, a typical feature for a ratchet-like mechanism. Except for Zn^2 +, the sugar puckering of the primer strand 3′ terminus changes from C2′-endo to C3′-endo during catalysis. In addition, our data are compatible with a scheme where metal A is the last component that binds to the active site to complete its productive assembly, as already inferred in human pol beta. The new structures have potential implications for modeling pol mu, a closely related polX implicated in the repair of DNA double-strand breaks, in a complex with a DNA synapsis.     

Relationship between phosphorylation of tyrosine aminotransferase and regulation of its synthesis by cyclic AMP and hormones.

Tyrosine adminotransferase (EC 2.6.1.5) has been found to be phosphorylated in intact rat hepatoma cells in culture. Incorporation of [32p]i into the enzyme is rapid and is exclusively found as phosphoserine. Cycloheximide treatment reduced phosphorylation of the aminotransferase only slightly and in the presence of three different inducers of this enzyme, dexamethasone, insulin, and dibutyryl cyclic AMP, [32P]I incorporation was increased. It is concluded that [32p]i incorporation into this enzyme probably reflects turnover of phosphate groups associated with pre-existing enzyme molecules catalyzed by a cyclic AMP-independent protein kinase.     

A soluble factor and GTP gamma S are required for Dictyostelium discoideum guanylate cyclase activity.

Amoeba of Dictyostelium discoideum show a rapid, transient cGMP synthesis in response to chemotactic stimulation. Using Mg(2+)-GTP as a substrate, guanylate cyclase (E.C. 4.6.1.2.) activity is found exclusively in the particulate fraction of Dictyostelium cells. Here we show that the activity is dependent on the presence of the non-hydrolysable GTP-analogue GTP gamma S, which itself is only a poor substrate for the enzyme under the prevailing conditions. Evidence is presented that a transient exposure of the enzyme to GTP gamma S is sufficient to constitutively activate the enzyme. GTP gamma S-dependent activity is found to require a factor that can be separated from the enzyme by washing the particulate fraction with low salt buffer. Addition of the soluble cell fraction to these washed membranes restores enzyme activity.     

Identification of a single and non-essential cysteine residue in dextransucrase of Leuconostoc mesenteroides NRRL B-512F

Amino acid analysis of purified dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F was carried out. The enzyme is virtually devoid of cysteine residue there being only one cysteine residue in the whole enzyme molecule comprising over 1500 amino acid residues. The enzyme is rich in acidic amino acid residues. The number of amino acid residues was calculated based on the molecular weight of 188,000 (Goyal and Katiyar 1994). Amino sugars were not found, implying that the enzyme is not a glycoprotein. It has been shown earlier that the cysteine residue in dextransucrase is not essential for enzyme activity (Goyal and Katiyar 1998). The presence of only one cysteine residue per enzyme molecule illustrates that its tertiary structure is solely dependent on other types of non-covalent interactions such as hydrogen bonding, ionic and nonpolar hydrophobic interactions.     

Purification and partial characterization of a methylglyoxal reductase from goat liver

An enzyme which catalyzes the reduction of methylglyoxal to lactaldehyde has been isolated and purified from goat liver to apparent homogeneity. NADH was found to be a better substrate than NADPH for methylglyoxal reduction. Stoichiometrically equivalent amounts of lactaldehyde and NAD are formed from methylglyoxal and NADH. Enzyme activity was located only in the soluble supernatant fractions of liver cells. Of the various carbonyl compounds tested, methylglyoxal was found to be the best substrate. The pH optimum of the enzyme was found to be 6.5, and Km for methylglyoxal was 0.4 mM. The molecular weight of the enzyme was found to be 89000 by gel filtration on a Sephadex G-200 column. Electrophoresis on sodium dodecyl sulfate-polyacrylamide gel revealed that the enzyme is composed of two subunits. The enzyme is highly sensitive to sulfhydryl group reagents. The inactivation by p-chloromercuribenzoate could be substantially protected by methylglyoxal in combination with NADH, indicating a possible involvement of one or more sulfhydryl group(s) at the active site of the enzyme.     

Oxidase reactions of tomato anionic peroxidase

Tomato (Lycopersicon esculentum Mill) anionic peroxidase was found to catalyze oxidase reactions with NADH, glutathione, dithiothreitol, oxaloacetate, and hydroquinone as substrates with a mean activity 30% that of horseradish peroxidase; this is in contrast to the negligible activity of the tomato enzyme as compared to the horseradish enzyme in catalyzing an indoleacetic acid-oxidase reaction with only Mn²⁺ and a phenol as cofactors. Substitution of Ce³⁺ for Mn²⁺ produced an 18-fold larger response with the tomato enzyme than with the horseradish enzyme, suggesting a significant difference in the autocatalytic indoleacetic acid-oxidase reactions with these two enzymes. In attempting to compare enzyme activities with 2,4-dichlorophenol as a cofactor, it was found that reaction rates increased exponentially with both increasing cofactor concentration and increasing enzyme concentration. While the former response may be analogous to allosteric control of enzyme activity, the latter response is contrary to the principle that reaction rate is proportional to enzyme concentration, and additionally makes any comparison of enzyme activity difficult.     

Enzymic unwinding of DNA. 2. Chain separation by an ATP-dependent DNA unwinding enzyme.

The DNA-stimulated ATPase characterized in the accompanying paper is shown to be a DNA unwinding enzyme. Substrates employed were DNA, RNA hybrid duplexes and DNA-DNA partial duplexes prepared by polymerization on fd phage single-stranded DNA template. The enzyme was found to denature these duplexes in an ATP-dependent reaction, without detectably degrading. EDTA, an inhibitor of the Mg2+-requiring ATPase, was found to prevent denaturation suggesting that dephosphorylation of the ATP and not only its presence is required. These results together with those from enzyme-DNA binding studies lead to ideas regarding the mode of enzymic action. It is proposed that the enzyme binds, in an initial step, to a single-stranded part of the DNA substrate molecule and that from here, energetically supported by ATP dephosphorylation, it invades double-stranded parts separating base-paired strands by processive, zipper-like action. It is further proposed that chain separation results from the combined action of several enzyme molecules and that a tendency of the enzyme to aggregate with itself reflects a tendency of the molecules to cooperate. Various functions are conceivable for the enzyme.     

A new enzyme that specifically inactivates apo-protein of pyridoxal enzymes

We found a new inactivating enzyme in small intestine and skeletal muscles, which specifically reacts with apo-proteins of pyridoxal enzymes. This inactivating enzyme does not react with all the non-pyridoxal enzymes tested. The inactivation by this enzyme is prevented by the addition of PALP. The enzyme splits apo-pyridoxal enzymes into smaller protein and oligopeptides. The activity of inactivating enzyme increases from 10–20 fold only in the case of B₆ deficiency compaired with that of normal rats.     

Characterization of an endoribonuclease, RNase N, from Escherichia coli.

The properties of the enzyme ribonuclease N were investigated. By comparing the distribution in the cell of RNase N with the bonafide intracellular beta-galactosidase, and the periplasmic alkaline phosphatase enzymes, we showed that RNase N is an intracellular enzyme. Since previous studies suggested that it is an endoribonuclease, it was compared to RNase III, the only other known intracellular endoribonuclease in Escherichia coli. Using homopolymers and co-polymers we found that, while RNase III could digest double-stranded RNA only, RNase N digested single-stranded and double-stranded RNA with similar efficiency. Furthermore, all RNAs used, natural as well as synthetic, were substrates for the enzyme. Using 5 S rRNA as a substrate it was confirmed that the enzyme is an endonuclease. The final products of the reaction of this enzyme are 5'-mononucleotides. The molecular weight of the enzyme is about 120,000 and it seems to contain two subunits which are similar in size. These properties thus differentiate this enzyme from all other known ribonucleases in E. coli.     

Association of the synaptic form of acetylcholinesterase with extracellular matrix in cultured mouse muscle cells

Myotubes of a mouse muscle-cell line (C2) synthesize in culture a 16S form of acetylcholinesterase that is normally found only in regions of adult mouse muscle that contain endplates. The 16S enzyme in C2 cell extracts has the properties expected of acetylcholinesterase forms that have a collagen-like tail. In intact cells, the active site of the 16S acetylcholinesterase is protected by a membrane-impermeable inhibitor, and this form of the enzyme can be removed by treatment of the cells with collagenase. Thus the enzyme is extracellular. Its extraction by high ionic strength solutions lacking detergent suggests that the 16S form is associated with the extracellular matrix by ionic interactions. Histochemical staining shows focal patches of acetylcholinesterase activity on the cell surface. Collagenase treatment, which removes only the 16S form, abolishes this staining pattern, indicating that the patches consist of the 16S enzyme. We conclude that the 16S enzyme in C2 myotubes occurs in focal patches on the cell surface, where it is associated with the extracellular matrix.