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PubMed1990

Sequence of choline acetyltransferase temperature-sensitive mutants determined by the polymerase chain reaction.

Two temperature-sensitive alleles of Drosophila melanogaster, Cha(ts1) and Cha(ts2), have been previously identified and are thought to be point mutations in the structural gene for the neurotransmitter biosynthetic enzyme choline acetyltransferase. In order to clarify the molecular nature of these alleles and characterize the presumed amino acid substitutions, we have used the polymerase chain reaction to amplify choline acetyltransferase messenger ribonucleic acid fragments from both mutant genotypes. Amplified mutant complementary deoxyribonucleic acid was cloned and used to construct chimeric complementary deoxyribonucleic acid clones containing approximately two-thirds of the wild-type sequence which would code for N-terminal amino acids and one-third of the mutant sequence coding for the C-terminal amino acids. After in vitro translation of complementary ribonucleic acid produced from the chimeric complementary deoxyribonucleic acid clones, choline acetyltransferase activity was determined and shown to be thermolabile. Sequence analyses of these clones showed that one amino acid substitution due to single base substitution is crucial in each chimeric choline acetyltransferase complementary deoxyribonucleic acid to generate a thermolabile choline acetyltransferase product. The point mutations of the structural gene for choline acetyltransferase are thus confirmed and shown to regulate the thermolability of the enzyme produced by Cha(ts1) and Cha(ts2).

Amino Acid Sequence↗
PubMedDate not supplied

Role of macrophages in the transmission of dengue virus-induced suppressor signal to a subpopulation of T lymphocytes.

In dengue type 2 virus (DV)-infected mice, the virus-specific immunosuppression is mediated by a two-step mechanism: (1) induction of T suppressor cells (Ts1) by by virus to produce a suppressor factor (SF) which (2) stimulates another subpopulation of T cells (Ts2) to produce prostaglandin which finally mediates suppression. SF suppresses DV-specific IgM plaque-forming cells (PFC) in the spleen cells sensitized in vivo or in vitro, as detected by Jerne's haemolytic plaque technique. The present study enabled us to investigate the role of an intermediary cell in transmission of the suppressor signal from Ts1 to Ts2. It was observed that SF was adsorbed on the surface of peritoneal macrophages. Live macrophages adsorbed SF, retrieved it from that adsorbed on heat-killed macrophages and presented it to the target cells. Heat-killed macrophages adsorbed SF to the same extent as live ones, but could present it to the target cells by themselves. The target cells of SF were unprimed splenic T lymphocytes. SF suppressed DV-specific PFC in syngeneic spleen cells and was adsorbed on syngeneic macrophages, but not on those from allogenic animals. The findings described here show that the presence of live macrophages is obligatory for transmission of the suppressor signal to the target Ts2.

Animals↗
PubMed1993

Secretion of bacterial beta-lactamase into culture broth by a mutant strain of Saccharomyces cerevisiae.

The secretion of Escherichia coli beta-lactamase was studied in a Saccharomyces cerevisiae ts1 mutant strain. The signal sequence of pre-beta-lactamase was recognized by the yeast cell and the precursor protein was processed to an enzymatically active mature beta-lactamase. In contrast to conventional strains, the mutant strain was able to secrete bla-gene-encoded beta-lactamase into the culture broth. These results indicate the potential usefulness of ts1 mutant strains for biotechnological purposes.

Biotechnology↗
PubMed2004

The merits of bipartite transition-state mimics for inhibition of uracil DNA glycosylase.

The glycosidic bond hydrolysis reaction of the enzyme uracil DNA glycosylase (UDG) occurs by a two-step mechanism involving complete bond breakage to the uracil anion leaving group in the first step, formation of a discrete glycosyl cation-uracil anion intermediate, followed by water attack in a second transition-state leading to the enzyme-bound products of uracil and abasic DNA. We have synthesized and determined the binding affinities of unimolecular mimics of the substrate and first transition-state (TS1) in which the uracil base is covalently attached to the sugar, and in addition, bimolecular mimics of the second addition transition state (TS2) in which the base and sugar are detached. We find that the bipartite mimics of TS2 are superior to the TS1 mimics. These results indicate that bipartite TS2 inhibitors could be useful for inhibition of glycosylases that proceed by stepwise reaction mechanisms.

Amides↗
PubMed2005

Effects of beta-adrenoceptor antagonists in the neural nitric oxide release induced by electrical field stimulation and sodium channel activators in the rabbit corpus cavernosum.

Beta-Adrenoceptor antagonists may present receptor-independent mechanisms, such as blockade of voltage-gated sodium channels. This study aimed to investigate the effects of non-selective (propranolol), and selective beta1- (atenolol, metoprolol and betaxolol) and beta2-adrenoceptor (ICI 118,551) antagonists in the nitric oxide (NO)-mediated rabbit corpus cavernosum relaxations induced by either electrical field stimulation (EFS) or activators of voltage-gated sodium channels. The sodium channel blockers tetrodotoxin and saxitoxin abolished the relaxations induced by EFS or sodium channel activators of binding site-2 (aconitine and veratridine), site-3 (Ts3 toxin), site-4 (Ts1 toxin) and site-5 (brevetoxin-3). The beta-adrenoceptor antagonists failed to affect the relaxations induced by EFS, aconitine and veratridine. Relaxations induced by Ts3 and Ts1 toxins, as well as brevetoxin-3, were markedly reduced by prior addition of propranolol, betaxolol and ICI 118,551. During the established relaxation induced by Ts3 toxin, propranolol failed to restore the basal tone. In conclusion, beta-adrenoceptor antagonists may cause an allosteric inhibition at the binding site-3, -4 and -5 of voltage-gated sodium channels, leading to blockade of neural NO release.

Aconitine↗
PubMed2005

In vivo clearing of idiotypic antibodies with antiidiotypic antibodies and their derivatives.

At immunolocalization of experimental tumors, idiotypic monoclonal antibodies, such as TS1 against cytokeratin 8, can be used to carry and deposit in vivo terapeutics in the tumor. These carriers also remain in the circulation and may cause negative side-effects in other tissues. In this report, several derivatives of the antiidiotypic antibody alphaTS1 were produced and tested for their clearing capacity of the idiotypic carrier antibody TS1. Intact monoclonal alphaTS1, scFv of a alphaTS1 and alphaTS1 Fab'2 and fragments were produced by recombinant technology or by cleavage with Ficin. The scFv was tailored by use of the variable domain genes of the light and heavy chain from the hybridoma clone in combination with a (Gly4Ser)3-linker, followed by expression in E. coli. When tested for clearing capacity, the intact divalent antiidiotypic IgG was found to be the most efficient. The divalent and the monovalent Fab fragment also demonstrated significant clearing, but lower than the intact antiidiotypic IgG. The alphaTS1 scFv antibody when injected separately was not found to clear the idiotype, but could do so when preincubated with the idiotype. Rapid excretion and in vivo instability of this low molecular weight antibody fragment may be the major reasons. Similar results were obtained when the system was reversed and the 131I-labeled antiidiotype IgG was cleared with the idiotype fragment. It is concluded that both intact antiidiotypic IgG, and Fab'2 fragments are able to clear the idiotypic antibodies. The experimental data support the conclusion that the Fc parts from both the idiotype and the antiidiotype may contribute to this elimination.

Antibodies, Anti-Idiotypic↗
PubMed2000

Modulation of G-protein linked cAMP accumulation in immortalized murine cortical astrocytes by retroviral infection.

The present study characterized beta-adrenergic receptors (beta-AR) in a clonal cell line (C1) immortalized from cerebral cortical astroglial cells of FVB/N mice. We also determined whether the wild type Moloney murine leukemia virus (wt-MoMuLV) and one of its neuropathogenic mutants, ts1-MoMuLV, modulated the beta-AR system in these cells. We observed that C1 cells possess a functional beta-AR system coupled to cAMP accumulation and capable of normal agonist-induced regulation (desensitization). Significant increases were observed in forskolin stimulated cAMP accumulation in C1 cells infected by wt MoMuLV and by ts1-MoMuLV. In contrast, the cAMP response to beta-AR stimulated by isoproterenol was relatively spared after viral exposure.

Adenylyl Cyclases↗
PubMed2000

Role of insulin receptor substrate-2 in interleukin-9-dependent proliferation.

Interleukin-9 (IL-9) stimulation results in JAK, STAT and IRS1/2 phosphorylation. The role of IRS adaptor proteins in IL-9 signaling is not clear. We show that IL-9 induces IRS2 phosphorylation and association with phosphatidylinositol-3 kinase (PI 3-K) p85 subunit in TS1 cells and BaF/9R cells, which proliferate upon IL-9 stimulation. We observed a PI 3-K-dependent phosphorylation of protein kinase B (PKB) in TS1 cells, but not in BaF/9R, nor in other IL-9-dependent cell lines. Finally, 32D cells that were transfected with the IL-9 receptor but lack IRS expression survived in the presence of IL-9. Ectopic IRS1 expression allowed for IL-9-induced proliferation, in the absence of significant PKB phosphorylation.

Animals↗
PubMed2003

Relaxation of rabbit corpus cavernosum by selective activators of voltage-gated sodium channels: role of nitric oxide-cyclic guanosine monophosphate pathway.

OBJECTIVES: To investigate the capacity of voltage-gated Na(+) channel activators such as batrachotoxin, aconitine, veratridine, Ts1 (formerly Tityus gamma-toxin), and brevetoxin-3 to induce relaxation of rabbit isolated corpus cavernosum (RbCC) and the pharmacologic mechanisms underlying this phenomenon. The voltage-gated Na(+) channels of the corpus cavernosum are essential for erectile function. A number of biologic toxins exert their effects by modifying the properties of these channels. METHODS: Male New Zealand white rabbits were anesthetized with pentobarbital sodium. Strips of RbCC were transferred to 10-mL organ baths containing oxygenated and warmed Krebs solution. The RbCC strips were connected to force-displacement transducers, and changes in isometric force were recorded using a PowerLab 400 data acquisition system. Corporeal smooth muscle was precontracted submaximally with phenylephrine (10 micromol/L). RESULTS: The binding site-2 (batrachotoxin, aconitine, and veratridine) and binding site-5 (brevetoxin-3) voltage-gated Na(+) channel activators caused slow-onset RbCC relaxations, and the binding site-4 activator Ts1 produced transitory relaxations followed by a return to baseline. The Na(+)channel blockers tetrodotoxin and saxitoxin (0.1 micromol/L each) abolished the relaxations induced by these agonists. Similarly, the nitric oxide synthase inhibitor N(omega)-nitro-l-arginine methyl ester (100 micromol/L) markedly reduced the relaxations and l-arginine (1 mmol/L) restored the relaxations. The soluble guanylyl cyclase inhibitor 1H-[1,2,4] oxidiazolo[4,3-alpha] quinoxalin-1-one (10 micromol/L) reduced the relaxations, and the phosphodiesterase type 5 inhibitor sildenafil (100 nmol/L) significantly potentiated the relaxations by all activators. CONCLUSIONS: Our results indicate that the relaxations evoked by selective activators of voltage-gated Na(+) channels are mediated by the release of nitric oxide from nitrergic nerves and the activation of the nitric oxide-cyclic guanosine monophosphate pathway in the smooth muscle cells of erectile tissue.

Aconitine↗
PubMed2002

Thermal and photochemical rearrangement of bicyclo[3.1.0]hex-3-en-2-one to the ketonic tautomer of phenol. Computational evidence for the formation of a diradical rather than a zwitterionic intermediate.

The ground state (S(0)) and lowest-energy triplet state (T(1)) potential energy surfaces (PESs) concerning the thermal and photochemical rearrangement of bicyclo[3.1.0]hex-3-en-2-one (8) to the ketonic tautomer of phenol (11) have been extensively explored using ab initio CASSCF and CASPT2 calculations with several basis sets. State T(1) is predicted to be a triplet pipi lying 66.5 kcal/mol above the energy of the S(0) state. On the S(0) PES, the rearrangement of 8 to 11 is predicted to occur via a two-step mechanism where the internal cyclopropane C-C bond is broken first through a high energy transition structure (TS1-S(0)()), leading to a singlet intermediate (10-S(0)()) lying 25.0 kcal/mol above the ground state of 8. Subsequently, this intermediate undergoes a 1,2-hydrogen shift to yield 11 by surmounting an energy barrier of only 2.7 kcal/mol at 0 K. The rate-determining step of the global rearrangement is the opening of the three-membered ring in 8, which involves an energy barrier of 41.2 kcal/mol at 0 K. This high energy barrier is consistent with the fact that the thermal rearrangement of umbellulone to thymol is carried out by heating at 280 degrees C. Regarding the photochemical rearangement, our results suggest that the most efficient route from the T(1) state of 8 to ground state 11 is the essentially barrierless cleavage of the internal cyclopropane C-C bond followed by radiationless decay to the S(0) state PES via intersystem crossing (ISC) at a crossing point (S(0)()/T(1)()-1) located at almost the same geometry as TS1-S(0)(), leading to the formation of 10-S(0)() and the subsequent low-barrier 1,2-hydrogen shift. The computed small spin-orbit coupling between the T(1) and S(0) PESs at S(0)()/T(1)()-1 (1.2 cm(-)(1)) suggests that the ISC between these PESs is the rate-determining step of the photochemical rearrangement 8 --> 11. Finally, computational evidence indicates that singlet intermediate 10-S(0)() should not be drawn as a zwitterion, but rather as a diradical having a polarized C=O bond.

Journal Article↗
PubMed2003

Mechanism of 1,3-migration in allylperoxyl radicals: computational evidence for the formation of a loosely bound radical-dioxygen complex.

The three pathways postulated for 1,3-migration of the peroxyl group in the allylperoxyl radical (1a), a key reaction involved in the spontaneous autoxidation of unsaturated lipids of biological importance, have been investigated by means of quantum mechanical electronic structure calculations. According to the barrier heights calculated from RCCSD(T)/6-311+G(3df,2p) energies with optimized molecular geometries and harmonic vibrational frequencies determined at the UMP2/6-311+G(3df,2p) level, the allylperoxyl rearrangement proceeds by fragmentation of 1a through a transition structure (TS1) with a calculated DeltaH++(298 K) of 21.7 kcal/mol to give an allyl radical-triplet dioxygen loosely bound complex (CX). In a subsequent step, the triplet dioxygen moiety of CX recombines at either end of the allyl radical moiety to convert the complex to the rearranged peroxyl radical (1a') or to revert to the starting peroxyl radical 1a. CX shows an electron charge transfer of 0.026 e in the direction allyl --> O(2). The dominant attractive interactions holding in association the allyl radical-triplet dioxygen pair in CX are due chiefly to dispersion forces. The DeltaH(298 K) for dissociation of CX in its isolated partners, allyl radical and triplet dioxygen, is predicted to be at least 1 kcal/mol. The formation of CX prevents the diffusion of its partners and maintains the stereocontrol along the fragmentation-recombination processes. The concerted 1,3-migration in allylperoxyl radical is predicted to take place through a five-membered ring peroxide transition structure (TS2) showing two long C-O bonds. The DeltaH++(298 K) calculated for this pathway is less favorable than the fragmentation-recombination pathway by 1.9 kcal/mol. The cyclization of 1a to give a dioxolanyl radical intermediate (2a) is found to proceed through a five-membered ring transition structure (TS3) with a calculated DeltaH++(298 K) of 33.9 kcal/mol. Thus, the sequence of ring closure 1a --> 2a and ring opening 2a --> 1a' is unlikely to play any significant role in allylperoxyl rearrangement 1a --> 1a'. In the three pathways investigated, the energy of the transition structure is predicted to be somewhat lower in either heptane or aqueous solution than in the gas phase. Although the energy lowering calculated for TS1 is smaller than the calculated for TS2 and TS3, it is very unlikely that the solvent effects may reverse the predicted preference of the fragmentation-recombination pathway over the concerted and stepwise ring closure-ring opening mechanisms.

Allyl Compounds↗
PubMed2005

New selective haloform-type reaction yielding 3-hydroxy-2,2-difluoroacids: theoretical study of the mechanism.

Experimental results of an unprecedented haloform-type reaction in which 4-alkyl-4-hydroxy-3,3-difluoromethyl trifluoromethyl ketones undergo base-promoted selective cleavage of the CO-CF(3) bond, yielding 3-hydroxy-2,2-difluoroacids and fluoroform, are rationalized using DFT (B3LYP) calculations. The gas-phase addition of hydroxide ion to 1,1,1,3,3-pentafluoro-4-hydroxypentan-2-one (R) is found to be a barrierless process, yielding a tetrahedral intermediate (INT), involving a DeltaG(r)(298 K) of -61.4 kcal/mol. The CO-CF(3) bond cleavage in INT leads to a hydrogen-bonded [CH(3)CHOHCF(2)CO(2)H...CF(3)](-) complex by passage through a transition structure (TS1) with a DeltaG()(298 K) of 20.8 kcal/mol and a DeltaG(r)(298 K) of 9.8 kcal/mol. This complex undergoes a proton transfer between its components, yielding a hydrogen-bonded [CH(3)CHOHCF(2)CO(2)...CHF(3)](-) complex. This process has associated with it a DeltaG()(298 K) of only 3.1 kcal/mol and a DeltaG(r)(298 K) of -43.3 kcal/mol. The CO-CF(2) bond cleavage in INT leads to a hydrogen-bonded [CH(3)CHOHCF(2)...CF(3)CO(2)H](-) complex by passage through a transition structure (TS3) with a DeltaG()(298 K) of 29.2 kcal/mol and a DeltaG(r)(298 K) of 25.1 kcal/mol. The lower energy barrier found for CO-CF(3) bond cleavage in INT is ascribed to the larger number of fluorine atoms stabilizing the negative charge accumulated on the CF(3) moiety of TS1, as compared to the number of fluorine atoms stabilizing the negative charge on the CH(3)CHOHCF(2) moiety of TS3. The solvent-induced effects on the two pathways, introduced within the SCRF formalism through PCM calculations, do not reverse the predicted preference of the CO-CF(3) over the CO-CF(2) bond cleavage of R in the gas phase.

Journal Article↗
PubMed1999

Preparation and Conversion of N-Halomethylpyridinium Halides. Comparison with Related Compounds.

N-Halomethylpyridinium halides 1a-f (X-CH(2)Py(+)X(-), X = Cl, Br) have been synthesized from a three-component reaction mixture containing a thionyl halide 5, formaldehyde (6), and a pyridine 7. The salts 1a-f react readily with a variety of heterocyclic nucleophiles to yield (in general, nonsymmetrical) 1,1-bis(heteroarylium)methyl salts 2ea-hb, (pathway a). The use of trichloroacetaldehyde (9) instead of formaldehyde in this three-component reaction leads to a salt 10 in which one of the CH(2)-hydrogens was replaced by the electron-withdrawing CCl(3) substituent. This changes the standard reaction pathway a of 1 in solution toward nucleophiles completely: the chlorinated N-vinylpyridinium salts 11 and 12 were formed after the reaction of 10 with pyridine or triphenylphosphane. These are useful intermediates for the synthesis of new N- and 4-substituted 1,4-dihydropyridines 13-15 as could be demonstrated for compound 11. To explain the reactivity pattern of compounds 1 and 10 and the related structures (MeO-CH(2)Py(+), 16, and Me(3)SiO-CH(2)Py(+), 17) we calculated, using ab initio and DFT methods, reaction pathways a and b, both in the gas phase and in solution using ammonia as a model nucleophile. For all of these compounds, pyridine displacement (pathway b) dominates in the gas phase. As an example the energy gap between these two transition states for 1a turns out to be relatively small (11.6 kcal/mol in favor of pathway b, TS2). Solvation effects can therefore stabilize the corresponding transition state TS1 more effectively. In a MeCN solution, TS1 is 1.6 kcal/mol less energetic than TS2.

Journal Article↗
PubMed2005

Modeling effects of oxyanion hole on the ester hydrolysis catalyzed by human cholinesterases.

Molecular dynamics (MD) simulations and hydrogen bonding energy (HBE) calculations have been performed on the prereactive enzyme-substrate complexes (ES), transition states (TS1), and intermediates (INT1) for acetylcholinesterase (AChE)-catalyzed hydrolysis of acetylcholine (ACh), butyrylcholinesterase (BChE)-catalyzed hydrolysis of ACh, and BChE-catalyzed hydrolysis of (+)/(-)-cocaine to examine the protein environmental effects on the catalytic reactions. The hydrogen bonding of cocaine with the oxyanion hole of BChE is found to be remarkably different from that of ACh with AChE/BChE. Whereas G121/G116, G122/G117, and A204/A199 of AChE/BChE all can form hydrogen bonds with ACh to stabilize the transition state during the ACh hydrolysis, BChE only uses G117 and A199 to form hydrogen bonds with cocaine. The change of the estimated total HBE from ES to TS1 is ca. -5.4/-4.4 kcal/mol for AChE/BChE-catalyzed hydrolysis of ACh and ca. -1.7/-0.8 kcal/mol for BChE-catalyzed hydrolysis of (+)/(-)-cocaine. The remarkable difference of approximately 3 to 5 kcal/mol reveals that the oxyanion hole of AChE/BChE can lower the energy barrier of the ACh hydrolysis significantly more than that of BChE for the cocaine hydrolysis. These results help to understand why the catalytic activity of AChE against ACh is considerably higher than that of BChE against cocaine and provides valuable clues on how to improve the catalytic activity of BChE against cocaine.

Acetylcholine↗
PubMed2006

Hydrogen transfer between sulfuric acid and hydroxyl radical in the gas phase: competition among hydrogen atom transfer, proton-coupled electron-transfer, and double proton transfer.

In an attempt to assess the potential role of the hydroxyl radical in the atmospheric degradation of sulfuric acid, the hydrogen transfer between H2SO4 and HO* in the gas phase has been investigated by means of DFT and quantum-mechanical electronic-structure calculations, as well as classical transition state theory computations. The first step of the H2SO4 + HO* reaction is the barrierless formation of a prereactive hydrogen-bonded complex (Cr1) lying 8.1 kcal mol(-1) below the sum of the (298 K) enthalpies of the reactants. After forming Cr1, a single hydrogen transfer from H2SO4 to HO* and a degenerate double hydrogen-exchange between H2SO4 and HO* may occur. The single hydrogen transfer, yielding HSO4* and H2O, can take place through three different transition structures, the two lowest energy ones (TS1 and TS2) corresponding to a proton-coupled electron-transfer mechanism, whereas the higher energy one (TS3) is associated with a hydrogen atom transfer mechanism. The double hydrogen-exchange, affording products identical to reactants, takes place through a transition structure (TS4) involving a double proton-transfer mechanism and is predicted to be the dominant pathway. A rate constant of 1.50 x 10(-14) cm(3) molecule(-1) s(-1) at 298 K is obtained for the overall reaction H2SO4 + HO*. The single hydrogen transfer through TS1, TS2, and TS3 contributes to the overall rate constant at 298 K with a 43.4%. It is concluded that the single hydrogen transfer from H2SO4 to HO* yielding HSO4* and H2O might well be a significant sink for gaseous sulfuric acid in the atmosphere.

Journal Article↗
PubMed1997

Mouse Moloney leukemia virus infects microglia but not neurons even though it induces motor neuron disease.

Motor neuron degeneration caused by ts1 MoMuLV occurs by an indirect mechanism and hypothetically appears associated with a two-cell or three-cell pathogenesis hypothesis. The first step in this hypothesis is associated with a small subset of resident microglial cells that serve as the principal target cells for ts1 MoMuLV infection. The second step is likely linked to trophic events, probably mediated by cytokines, that lead to hypertrophy and activation of a substantial number of additional microglial cells (autocrine effect) and adjacent astrocytes (paracrine effect). The third step in this hypothesis appears related to indirect neuronal degeneration mediated by cytotoxins produced by activated microglial cells and astrocytes. In this last step, motor neurons located within these foci of activated microglial cells and astrocytes are 'innocent bystander cells' and degenerate and die due to paracrine effects. The mechanism of motor neuron degeneration is poorly understood but is likely linked to a sequential cascade of trophic factors and cytokines resulting in a final common pathway for motor neuron death involving production of oxidative radicals, excitatory aminoacid neurotransmitter-like substances, prostaglandins, or nitric oxide.

Animals↗
PubMed2005

Imidoylketene-alpha-oxoketenimine and alpha-oxoketene-alpha-oxoketene rearrangements. 1,3-shifts of substituted phenyl groups.

1,3-phenyl shifts interconvert imidoylketenes 1 and alpha-oxoketenimines 2 and, likewise, alpha-oxoketenes 3 automerize by this 1,3-shift. These rearrangements usually take place in the gas phase under conditions of flash vacuum thermolysis. Energy profiles calculated at the B3LYP/6-31G(d,p) and B3LYP/6311 + G(3df,2p)//B3LYP/6-31G(d,p) levels demonstrate that electron donating substituents (D) in the migrating phenyl group and electron withdrawing ones (W) in the non-migrating phenyl group, can stabilise the transition states TS1 and TS2 to the extent that activation barriers of ca. 100 kJ mol(-1) or less are obtained; i.e. enough to make these reactions potentially observable in solution at ordinary temperatures. The calculated transition state energies DeltaG(TS1) show an excellent correlation with the Hammett constants sigma(p)(W) and sigma(p+)(D).

Journal Article↗
PubMed1998

Mutant DNA polymerase delta from thermosensitive Schizosaccharomyces pombe strains display reduced stimulation by proliferating cell nuclear antigen.

We have isolated and characterized DNA polymerase delta (pol delta) from two thermosensitive Schizosaccharomyces pombe strains, poldeltats1 and poldeltats3, mutated in two different evolutionarily conserved domains of the catalytic subunit. At the restrictive temperature of 37 degreesC poldeltats1 and poldeltats3 mutant strains arrest growth in the S phase of the cell cycle. We show that at low levels of primer ends, in vitro stimulation by proliferating cell nuclear antigen (PCNA) of mutant enzymes is lower than stimulation of wild-type pol delta. Affinity for primer (3'-OH) ends and processivity of mutant enzymes do not appear different from wild-type pol delta. In contrast, Vmax values are lower than the wild-type value. The major in vitro defect appears to be decreased stimulation of mutant enzymes by PCNA, resulting in reduced velocity of DNA synthesis. In addition, ts1 pol delta is not stimulated by low PCNA concentration at 37 degreesC, although low concentrations stimulate activity at 25 degreesC, suggesting that this thermolability at low levels of primer ends could be its critical defect in vivo. Thus, both ts1 and ts3 pol delta mutations are located in regions of the catalytic subunit that seem necessary, directly or indirectly, for its efficient interaction with PCNA.

Amino Acid Sequence↗