A genetic system to identify DNA polymerase beta mutator mutants

Hemagglutinins were determined in six species of mosquitoes that are susceptible and refractory to Brugia malayi (Filarioidea: Nematoda). High titers of hemagglutinins were found in the salivary gland extract and in the body fluid of a completely refractory species, Aedes taeniorhynchus, and in partially refractory species, Anopheles quadrimculatus but low levels of hemagglutinins were also present in the body fluid of Aedes aegypti (Black-eye, Liverpool strain), a susceptible species. Hemagglutinating activity was not found in the other three completely refractory species of mosquitoes, Culex quinquefasciatus, Culex nigripalpus, and Aedes albopictus in which blood coagulated rapidly after ingestion. High titers of hemagglutinins in the salivary glands of Ae. taeniorhynchus and An. quadrimaculatus facilitated rapid movement of sheathed microfilariae from the midgut to the hemocoel. It is suggested that high titers of hemagglutinins present in the hemocoel bound to the glycoconjugates with exposed carbohydrate moieties present on the microfilarial sheaths and developing abnormal larvae (L1) in the thoracic muscle cells. These hemagglutinin-bound glycoconjugates formed capsules that subsequently stimulated the immune response and resulted in melanization of microfilarial sheaths and sheathed microfilariae in the hemocoel and intracellularly developing abnormal L1 in the thoracic muscles. Only minimal encapsulation and melanization of B. malayi microfilariae was observed in the hemocoel of the other four species of mosquitoes that lacked hemagglutinins in the salivary glands. The results suggest that tissue specific hemagglutinins are one of several factors of vector susceptibility/refractoriness through immune reactions (encapsulation, activation of prophenoloxidases).     

Detection of a mutator phenotype in cancer cells by inter-Alu polymerase chain reaction

A mutator phenotype due to a DNA mismatch repair deficiency is usually detected by typing a number of microsatellite markets. Here, eight hereditary nonpolyposis colon cancer patients with microsatellite instability were investigated by inter-Alu PCR, known to amplify DNA segments that may represent preferential targets of replication errors. Among 40-60 bands revealed in a single PCR experiment, more than 20% were found altered in tumoral DNA samples compared to matched normal samples from the same patient. Shifts and changes in signal intensity accounted for most of the alterations, whereas gains or losses of bands were rare. Certain bands were affected only in a single patient, whereas the instabilities in others were common. These results suggest that some genomic regions are more susceptible than others to the expression of a mutator phenotype. Four such bands altered in at least five patients were characterized further and shown to be unstable because of contractions of the Alu poly(A) tails. Interestingly, none of the bands representing loci shown previously to be polymorphic in the population displayed instability in the tumoral samples. Inter-Alu PCR appears to be a robust, cost-effective, and sensitive technique for revealing the mutator phenotype in cancer cells.     

Overexpression of transforming growth factor beta type I receptor abolishes malignant phenotype of a rat bladder carcinoma cell line

The disposition of ISIS 2922, a phosphorothioate oligonucleotide for treatment of cytomegalovirus associated retinitis, was evaluated in rabbits. Vitreous humor and retina samples were collected from rabbits that received a single intravitreal injection of 66 microg [14C]-labeled ISIS 2922 and were analyzed using anion exchange HPLC. Four hr postdosing, the concentration of ISIS 2922 in vitreous humor was 3.3 microM. The elimination of ISIS 2922 from the vitreous humor exhibited first-order kinetics with a t1/2 of 62 hr. By 10 days postdosing, the mean concentration of ISIS 2922 in rabbit vitreous humor had decreased to 0.17 microM, which represented 22% of the total radioactivity remaining in the vitreous. The remaining 78% coeluted on anion exchange HPLC with shorter oligonucleotides. In retina, ISIS 2922 accumulated over the first 5 days postdosing, reaching a maximum concentration of 3.5 microM, and then declined thereafter with an estimated t1/2 of 79 hr. By 10 days postdosing when only 24% of the total radioactivity in the retina was parent compound, the concentration of ISIS 2922 remained at 1.6 microM, which was 10 times higher than the concentration in the vitreous humor. Whereas the elimination of full-length ISIS 2922 and total radioactivity from the vitreous humor occurred at nearly equal rates, ISIS 2922 disappeared more rapidly than did total radioactivity from the retina, suggesting a greater role for metabolism in the clearance process from retina than the vitreous. Alternatively, the results are consistent with metabolites being cleared from the vitreous at approximately the same rate as parent compound while in the retina metabolites may be cleared more slowly. The data were analyzed with a user-defined pharmacokinetic model, which was then used to predict the potential for accumulation of ISIS 2922 during clinical dosing.     

Examination of the role of DNA polymerase proofreading in the mutator effect of miscoding tRNAs

We previously described Escherichia coli mutator tRNAs that insert glycine in place of aspartic acid and postulated that the elevated mutation rate results from generating a mutator polymerase. We suggested that the proofreading subunit of polymerase III, epsilon, is a likely target for the aspartic acid-to-glycine change that leads to a lowered fidelity of replication, since the altered epsilon subunits resulting from this substitution (approximately 1% of the time) are sufficient to create a mutator effect, based on several observations of mutD alleles. In the present work, we extended the study of specific mutD alleles and constructed 16 altered mutD genes by replacing each aspartic acid codon, in series, with a glycine codon in the dnaQ gene that encodes epsilon. We show that three of these genes confer a strong mutator effect. We have also looked for new mutator tRNAs and have found one: a glycine tRNA that inserts glycine at histidine codons. We then replaced each of the seven histidine codons in the mutD gene with glycine codons and found that in two cases, a strong mutator phenotype results. These findings are consistent with the epsilon subunit playing a major role in the mutator effect of misreading tRNAs.     

The fidelity of DNA polymerase beta during distributive and processive DNA synthesis

During base excision repair, DNA polymerase beta fills 1-6-nucleotide gaps processively, reflecting a contribution of both its 8- and 31-kDa domains to DNA binding. Here we report the fidelity of pol beta during synthesis to fill gaps of 1, 5, 6, or >300 nucleotides. Error rates during distributive synthesis by recombinant rat and human polymerase (pol) beta with a 390-base gap are similar to each other and to previous values with pol beta purified from tissues. The base substitution fidelity of human pol beta when processively filling a 5-nucleotide gap is similar to that with a 361-nucleotide gap, but "closely-spaced" substitutions are produced at a rate at least 60-fold higher than for distributive synthesis. Base substitution fidelity when filling a 1-nucleotide gap is higher than when filling a 5-nucleotide gap, suggesting a contribution of the 8-kDa domain to the dNTP binding pocket and/or a difference in base stacking or DNA structure imposed by pol beta. Nonetheless, 1-nucleotide gap filling is inaccurate, even generating complex substitution-addition errors. Finally, the single-base deletion error rate during processive synthesis to fill a 6-nucleotide gap is indistinguishable from that of distributive synthesis to fill a 390-nucleotide gap. Thus the mechanism of processivity by pol beta does not allow the enzyme to suppress template misalignments.     

Increased activity and fidelity of DNA polymerase beta on single-nucleotide gapped DNA

DNA polymerase beta (pol beta) is an error-prone polymerase that plays a central role in mammalian base excision repair. To better characterize the mechanisms governing rat pol beta activity, we examined polymerization on synthetic primer-templates of different structure. Steady-state kinetic analyses revealed that the catalytic efficiency of pol beta (kcat/Km,dNTPapp) is strongly influenced by gap size and the presence of a phosphate group at the 5'-margin of the gap. pol beta exhibited the highest catalytic efficiency on 5'-phosphorylated 1-nucleotide gapped DNA. This efficiency was >/=500 times higher than on non-phosphorylated 1-nucleotide and 6-nucleotide (with or without PO4) gapped DNAs and 2,500 times higher than on primer-template with no gaps. The nucleotide insertion fidelity of pol beta, as judged by its ability to form G-N mispairs, was also higher (10-100 times) on 5'-phosphorylated single-nucleotide gapped DNA compared with the other DNA substrates studied. These data suggest that a primary function of mammalian pol beta is to fill 5'-phosphorylated 1-nucleotide gaps.     

The Pol beta-14 dominant negative rat DNA polymerase beta mutator mutant commits errors during the gap-filling step of base excision repair in Saccharomyces cerevisiae

We demonstrated recently that dominant negative mutants of rat DNA polymerase beta (Pol beta) interfere with repair of alkylation damage in Saccharomyces cerevisiae. To identify the alkylation repair pathway that is disrupted by the Pol beta dominant negative mutants, we studied the epistatic relationship of the dominant negative Pol beta mutants to genes known to be involved in repair of DNA alkylation damage in S. cerevisiae. We demonstrate that the rat Pol beta mutants interfere with the base excision repair pathway in S. cerevisiae. In addition, expression of one of the Pol beta dominant negative mutants, Pol beta-14, increases the spontaneous mutation rate of S. cerevisiae whereas expression of another Pol beta dominant negative mutant, Pol beta-TR, does not. Expression of the Pol beta-14 mutant in cells lacking APN1 activity does not result in an increase in the spontaneous mutation rate. These results suggest that gaps are required for mutagenesis to occur in the presence of Pol beta-14 but that it is not merely the presence of a gap that results in mutagenesis. Our results suggest that mutagenesis can occur during the gap-filling step of base excision repair in vivo.     

Purification and characterization of a new DNA polymerase from budding yeast Saccharomyces cerevisiae. A probable homolog of mammalian DNA polymerase beta

A new DNA polymerase activity was identified and purified to near homogeneity from extracts of mitotic and meiotic cells of the yeast Saccharomyces cerevisiae. This activity increased at least 5-fold during meiosis, and it was shown to be associated with a 68-kDa polypeptide as determined by SDS-polyacrylamide gel electrophoresis. This new DNA polymerase did not have any detectable 3'-->5' exonuclease activity and preferred small gapped DNA as a template-primer. The activity was inhibited by dideoxyribonucleoside 5'-triphosphates and N-ethylmaleimide but not by concentrations of aphidicolin which completely inhibit either DNA polymerases I (alpha), II (epsilon), or III (delta). Since no polypeptide(s) in the extensively purified DNA polymerase fractions cross-reacted with antibodies raised against yeast DNA polymerases I, II, and III, we called this enzyme DNA polymerase IV. The DNA polymerase IV activity increased at least 10-fold in a yeast strain overexpressing the gene product predicted from the YCR14C open-reading frame (identified on S. cerevisiae chromosome III and provisionally called POLX), while no activity was detected in a strain where POLX was deleted. These results strongly suggest that DNA polymerase IV is encoded by the POLX gene and is a probable homolog of mammalian DNA polymerase beta.     

Thermodynamics of human DNA ligase I trimerization and association with DNA polymerase beta

The interaction between human DNA polymerase beta (pol beta) and DNA ligase I, which appear to be responsible for the gap filling and nick ligation steps in short patch or simple base excision repair, has been examined by affinity chromatography and analytical ultracentrifugation. Domain mapping studies revealed that complex formation is mediated through the non-catalytic N-terminal domain of DNA ligase I and the N-terminal 8-kDa domain of pol beta that interacts with the DNA template and excises 5'-deoxyribose phosphate residue. Intact pol beta, a 39-kDa bi-domain enzyme, undergoes indefinite self-association, forming oligomers of many sizes. The binding sites for self-association reside within the C-terminal 31-kDa domain. DNA ligase I undergoes self-association to form a homotrimer. At temperatures over 18 degreesC, three pol beta monomers attached to the DNA ligase I trimer, forming a stable heterohexamer. In contrast, at lower temperatures (<18 degreesC), pol beta and DNA ligase I formed a stable 1:1 binary complex only. In agreement with the domain mapping studies, the 8-kDa domain of pol beta interacted with DNA ligase I, forming a stable 3:3 complex with DNA ligase I at all temperatures, whereas the 31-kDa domain of pol beta did not. Our results indicate that the association between pol beta and DNA ligase I involves both electrostatic binding and an entropy-driven process. Electrostatic binding dominates the interaction mediated by the 8-kDa domain of pol beta, whereas the entropy-driven aspect of interprotein binding appears to be contributed by the 31-kDa domain.     

Overexpression of superoxide dismutase in Trypanosoma cruzi results in increased sensitivity to the trypanocidal agents gentian violet and benznidazole

The proprioceptive feedback associated with the performance of even quite simple movements is always generated by the whole set of muscles subjected to mechanical deformation (lengthening, shortening, contraction, etc.) during that particular movement. The question was addressed here as to how muscle spindle feedbacks arising from agonist and antagonist muscles may contribute to the coding of movement parameters such as the direction and velocity. For this purpose, the activity of single muscle spindle afferents located in the lateral peroneal nerve was analysed using the microneurographic technique, in human subjects performing repetitive voluntary movements, i.e., plantar/dorsal flexions of the ankle, at three different velocities (3, 4.5 and 6 degrees/s). The data obtained suggest that in humans, the direction of a slow movement may be specified on the basis of the spindle discharge rate, which is greater in the stretched than in the shortened muscle, and that the velocity of this movement might be correlated with the difference between the spindle activity occurring in the agonist and antagonist muscles. These neurophysiological data are in agreement with the results of previous psychophysical studies showing for example that a sensation of illusory movement can be elicited only when there exists an imbalance between the agonist versus antagonist vibration-induced Ia inputs. In addition, the greater the difference between the vibration frequencies applied to the two antagonist muscles, the higher the perceived movement velocity was found to be. All in all, joint movement perception seems to result from the co-processing by the central nervous system of the multiple spindle feedbacks originating from the whole set of muscles involved in the performance of a movement.     

Preferential formation and decreased removal of cisplatin-DNA adducts in Chinese hamster ovary cell mitochondrial DNA as compared to nuclear DNA

We report here evaluation of a competitive enzyme-linked immunosorbent assay (c-ELISA) for detection of Salmonella spp. in chicken organs and faeces. The c-ELISA used a monoclonal antibody (MAb), specific for a genus-specific epitope of the outer core oligosaccharide of salmonellae. Salmonella lipopolysaccharide (LPS) in samples competed with Salmonella LPS coated on microtitre plates, for binding to the MAb. Competition reduced binding of the MAb to the LPS on the plate and of the secondary antibody to the MAb hence reducing the chromogenic signal. Stable coating and minimal false positive were achieved by conjugating LPS to poly-L-lysine. The c-ELISA was compared with motility enrichment culture using modified semisolid Rappaport Vassiliadis (MSRV) medium, which detected less than 10(2) CFU/g, and did not allow migration of non-salmonella species. The c-ELISA detected 10(6) CFU of enriched culture or 10(2)-10(3) CFU of Salmonella/g of faeces. Its limit of detection was thus higher than that of MSRV culture and it had a sensitivity of 92.9% and a specificity of 96.7%.     

Sensitivity of a mutator gene in Chinese hamster ovary cell to deoxynucleoside triphosphate pool alterations

The Thy- mutants of Chinese hamster ovary cells have a 5- to 10-fold elevated pool of deoxycytidine 5'-triphosphate (dCTP) and are auxotrophic for thymidine as an apparent consequence of a single mutation. thy is also a mutator gene, elevating the spontaneous rate of mutation 5- to 200-fold for at least two genetic markers. Previous experiments suggested that this mutator activity was caused by the elevated pool of dCTP in Thy- cells. To test this, the dCTP and deoxythymidine 5'-triphosphate (dTTP) pools were manipulated by altering the external concentration of thymidine in the growth medium. The rate of mutation at one genetic locus, ouabain resistance, was directly related to cellular dCTP content. At the highest level of dCTP the rate in one Thy- strain was approximately 200 times that of wild-type cells. However, the relationship between dCTP content and the rate of mutation at the ouabain locus was different for two mutator strains and wild-type cells. The rate of mutation at a second locus, thioguanine resistance, was increased approximately 10-fold over wild type regardless of the dCTP-dTTP pools. These experiments suggest that the mutator activity of thy is clearly related to dCTP content, but the dCTP level alone does not appear to be the cause of the mutator.     

Suppressors of the temperature sensitivity of DNA polymerase alpha mutations in Saccharomyces cerevisiae

We recently showed the involvement of the L-arginine/nitric oxide (NO) pathway in acid-induced duodenal mucosal bicarbonate secretion in rats. The aim of the present study was to confirm this observation in pigs by direct measurements of NO production. Experiments were performed on 16 anaesthetized pigs of both sexes treated with guanethidine (6 mg kg-1, intravenously). A duodenal segment, devoid of pancreaticobiliary influxes, was perfused with saline and the duodenal mucosal bicarbonate secretion was calculated from continuous measurements of pH and PCO2. The perfusate contents of NO and its oxidative product nitrite were determined by chemiluminescence, after reduction of nitrite to NO. Luminal acidification with 30 mM hydrochloric acid increased the output of bicarbonate as well as NO to the perfusate, by 195 +/- 45% and 106 +/- 10%, respectively. These responses to acid were markedly inhibited by adding the NO synthase inhibitor NG-monomethyl-L-arginine (L-NMMA, 0.3 mM) to the perfusate. The inhibitory effect of L-NMMA could be reversed by administration of L-arginine (3 mM). The study presents simultaneous measurements of bicarbonate and NO outputs to a duodenal luminal perfusate. The results strongly support the view that the L-arginine/NO pathway is involved in the acid-induced duodenal mucosal bicarbonate secretory response.     

Isolation of DNA polymerase beta from human placenta and study of its substrate specificity

A method of purification of DNA polymerase beta with a specific activity of 1300 units/mg from human placenta was developed. The enzyme preparations do not contain any other DNA polymerase activities and any nuclease contaminations degrading nucleic acids. On the basis of analysis of several standard parameters we conclude that the purified enzyme is polymerase beta. The optimal conditions of polymerization were established, and a comparison of the relative rates of polymerization with various template-primer complexes was carried out. Activated DNA was shown to be the optimal substrate in the presence of MgCl2, and poly(dA).oligo(dT) in the presence of MnCl2. The activation energies of polymerization for different template-primers were estimated.     

NMR spectroscopy as a tool to investigate the structural basis of anticancer drugs

NMR spectroscopy has been shown to be useful in determining the structures of nucleic acid fragments in solution. Over the last several years NMR spectroscopy, in conjunction with restrained molecular dynamics, has been employed to understand the 3D structures of a number of anticancer drugs and to rationalize their DNA binding behavior. In this review we address the methodologies used most frequently to determine nucleic acid structures in solution. In subsequent sections, we examine how these methods have been applied to rationalize the activities of a number of anticancer agents that target duplex DNA such as cisplatin, bleomycin and calicheamicin. Non-duplex DNA and RNA also represent interesting nucleic acid targets for anticancer drug design and applications of solution NMR spectroscopy to understanding the structures of these types of molecules (e.g. Okazaki fragments, DNA tetraplexes) are also reviewed. In the final sections, advances in NMR methodologies (e.g. linear prediction, superconducting probes) that are likely to impact the research conducted in this area are reviewed. The success of NMR spectroscopy in understanding the structural basis for clinically useful anticancer drugs bodes well for future applications of this methodology not only in rationalization of existing biological activity, but in the design of novel agents that will be useful in treating neoplastic disease.     

DNA polymerase beta: pre-steady-state kinetic analysis and roles of arginine-283 in catalysis and fidelity

DNA polymerase beta (pol beta) is the smallest and least complex DNA polymerase. The structure of the enzyme is well understood, but little is known about its catalytic properties, particularly processivity and fidelity. Pre-steady-state analysis of the incorporation of a single nucleotide into a short 25/45 oligonucleotide primer-template by pol beta was used to define the kinetic parameters of the polymerase. In addition, nucleotide analogs and site-specific mutants, along with structural analyses, were used to probe the structure-function relationship of pol beta. Several significant findings have been obtained: (i) The catalysis by pol beta is processive and displays an initial burst under pre-steady-state conditions, but the processivity is poor compared to other polymerases. (ii) The fidelity of pol beta is also low relative to other polymerases. (iii) Under pre-steady-state conditions the chemical step appears to be only partially rate-limiting on the basis of the low thio effect (4.3), defined as kpol(dNTP)/kpol(dNTP alpha S). The thio effect increases to 9 for incorporation of an incorrect nucleotide. These results are consistent with the existence of a substrate-induced conformational change that is also partially rate-limiting. (iv) A comparison between the two-dimensional NMR spectra of the wild-type and mutant enzymes indicates that the mutations at position 283 did not significantly perturb the structure of the enzyme. The conformational stability of the mutants is also unperturbed. Thus, R283 is not important to the overall structure of the enzyme. (v) The results of kinetic analyses of R283A and R283K mutants indicate that the hydrogen bond between R283 of pol beta and the template is important for catalysis. Both R283A and R283K mutants displayed decreases in catalytic efficiency by a factor of ca. 200 relative to wild-type pol beta. The mutants are also less faithful by a factor of 2-4, in terms of the T-G mispair vs the T-A correct pair. The perturbation, however, could occur at both the implied conformational step and the chemical step, since the thio effects of the mutants for both correct and incorrect nucleotides are similar to those of WT pol beta.     

The mutator form of polymerase beta with amino acid substitution at tyrosine 265 in the hinge region displays an increase in both base substitution and frame shift errors

This study describes the first complete in vitro error specificity analysis of a mutator DNA polymerase that is altered in a residue not predicted to contact either the DNA or dNTP substrate. We examined this mutator form of polymerase beta (Y265C) in order to elucidate the critical role tyrosine 265 plays in the accuracy of DNA synthesis. Our results demonstrate that an increase in both frame shift errors in homonucleotide repeat sequences and base substitution errors contribute nearly equally to the Y265C mutator phenotype. The models described for production of these errors, primer/template misalignment and base misincorporation, respectively, are distinctly different, suggesting the Y265C alteration affects discrimination against both types of error production pathways. In addition, Y265C displays a 530-fold increase in multiple errors within the 203-base pair target region examined, relative to that of wild type. Processivity studies revealed that Y265C retains the near distributive nature of DNA synthesis characteristic of the wild type polymerase beta. Therefore, multiple errors exhibited by Y265C most likely result from independent polymerase binding events. Localization of tyrosine 265 in the X-ray crystallographic structure suggests this residue may play a role in mediating a conformational change of the polymerase [Pelletier, H., et al. (1996) Biochemistry 35, 12742-12761]. A conformational change is predicted to enhance the accuracy of DNA synthesis by imposing an induced fit selection against premutational intermediates. The observed loss of discrimination against both misalignment-mediated and misincorporation-mediated errors produced by polymerase Y265C is consistent with such a model.