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At least 127 records · Page 7Linked to original sources
PubMed2001

A zinc ribbon protein in DNA replication: primer synthesis and macromolecular interactions by the bacteriophage T4 primase.

The gene product 61 primase protein from bacteriophage T4 was expressed as an intein fusion and purified to homogeneity. The primase binds one zinc ion, which is coordinated by four cysteine residues to form a zinc ribbon motif. Factors that influence the rate of priming were investigated, and a physiologically relevant priming rate of approximately 1 primer per second per primosome was achieved. Primase binding to the single-stranded binding protein (1 primase:4 gp32 monomers; K(d) approximately 860 nM) and to the helicase protein in the presence of DNA and ATP-gamma-S (1 primase:1 helicase monomer; K(d) approximately 100 nM) was investigated by isothermal titration calorimetry (ITC). Because the helicase is hexameric, the inferred stoichiometry of primase binding as part of the primosome is helicase hexamer:primase in a ratio of 1:6, suggesting that the active primase, like the helicase, might have a ring-like structure. The primase is a monomer in solution but binds to single-stranded DNA (ssDNA) primarily as a trimer (K(d) approximately 50-100 nM) as demonstrated by ITC and chemical cross-linking. Magnesium is required for primase-ssDNA binding. The minimum length of ssDNA required for stable binding is 22-24 bases, although cross-linking reveals transient interactions on oligonucleotides as short as 8 bases. The association is endothermic at physiologically relevant temperatures, which suggests an overall gain in entropy upon binding. Some possible sources of this gain in entropy are discussed.

Bacteriophage T4↗
PubMedDate not supplied

[Recognition of T-T cyclobutane pyrimidine dimers in the form of primers by DNA polymerases].

A study was made of the efficiency of primer conversion catalyzed by human placenta DNA polymerase on the length of primer. The dependence of -log Kd and Vmax on the number of mononucleotide units (n) of primer were shown to be linear up to n <--> 10. Each mononucleotide unit of the primer enhanced its affinity by a factor of 2.3 and the maximal polymerization rate by a factor of 1.3. For the first time estimation was made for the contribution of replication enzymes: human placenta DNA polymerase and E. coli DNA polymerase I (as well as AMV reverse transcriptase) to a decrease in the amount of T-T dimers in DNA emerging after UV- or gamma-irradiation. The efficiency of elongation of the d(pT)10 primer was shown to decrease with an increase in T-T dimers in the primer. When the d(pT)10 primer contains about 2.6 T-T dimers per molecule, the efficiency of its elongation decreases by a factor of 8-18.

DNA Primers↗
PubMedDate not supplied

Molecular detection of Burkholderia cepacia in toiletry, cosmetic, and pharmaceutical raw materials and finished products.

A polymerase chain reaction (PCR) assay was developed and compared with standard methods for rapid detection of Burkholderia cepacia, a major industrial contaminant, in cosmetic and pharmaceutical raw materials and finished products. Artificially contaminated samples were incubated for 24 h in trypticase soy broth containing 4% Tween 20 and 0.5% soy lecithin. DNA was extracted from each sample using a proteinase K-tris-EDTA-Tween 20 treatment at 35 degrees C. The extracted DNA was added to Ready-To-Go PCR beads and specific DNA primers for B. cepacia. The B. cepacia DNA primers coded for a 209-base pair (bp) fragment of the 16S rRNA ribosomal gene. No DNA amplification was observed in samples that were not spiked with B. cepacia. However, all contaminated samples showed the specific 209-bp fragment for B. cepacia. There was a 100% correlation between standard methods and the PCR assay. Standard microbiological methods required 5-6 days for isolation and identification of spiked microorganisms, whereas PCR detection and identification was completed in 27 h. PCR detection of B. cepacia allows for rapid quality evaluation of cosmetic and pharmaceutical raw materials and finished products.

Burkholderia cepacia↗
PubMed1966

Deoxyribonucleic acid nucleotidyltransferase from Landschütz ascites-tumour cells.

1. A purification procedure for DNA nucleotidyltransferase from Landschütz ascites-tumour cells is described. The enzyme can be separated from endogenous nucleic acid and from triphosphatase and deoxyribonuclease activities measurable at pH7.5. 2. The basic properties of the nucleotidyltransferase reaction are as follows. The enzyme has optimum activity at pH7.2-7.4. It displays an absolute requirement for DNA-primer, thermally-denatured DNA serving three to ten times as efficiently in this respect as native DNA. Maximum synthesis of polydeoxyribonucleotide occurs in the presence of all four deoxyribonucleoside 5'-triphosphates, but a limited incorporation of mononucleotide into polynucleotide is observed when the system is provided with only one triphosphate, or with various combinations of mono-, di- and tri-phosphates. The reaction requires the presence of a bivalent cation, and of those tested, Mg(2+) ions were by far the most effective. Manganous ions promoted synthesis but to a much smaller extent. Calcium ions did not support synthesis at all. At the appropriate concentrations, the univalent cations (sodium and potassium) stimulated the reaction by 25% and 125% respectively. The presence of EDTA in the reaction mixture stimulates the system five- to ten-fold. 3. The storage characteristics of the enzyme (as well as the activities of the various fractions) improve markedly if EDTA and 2-mercaptoethanol are included in the enzyme solution and in all preparative buffer solutions. 4. The enzyme loses more than 95% of its activity after heating for 1min. at 45 degrees . If the heating is conducted in the presence of DNA, the enzyme becomes relatively heat-resistant (presumably as a consequence of complex-formation with the DNA) and may actually display an activation effect. This is discussed in relation to a possible molecular conformation of the enzyme. 5. The product of the nucleotidyltransferase reaction is precipitable by acid or ethanol, and is susceptible to the actions of deoxyribonucleases I and II, snake-venom and spleen phosphodiesterases, and micrococcal nuclease. It forms a band in a density gradient of caesium chloride at a density similar to that of the DNA-primer. 6. By the criteria of nearest-neighbour frequency analyses, the product of the nucleotidyltransferase reaction has the characteristics to be expected of a polynucleotide synthesized in accordance with the template directions of the primer.

Chemical Phenomena↗
PubMed1997

Cloning and assembly of PCR products using modified primers and DNA repair enzymes.

We present a method for the creation of ligatable 3' overhangs by the incorporation of a modified base, uracil, at a specific position in the PCR primer and subsequent treatment with the DNA-modifying enzyme uracil DNA glycosylase and then either T4 endonuclease V or human apurinic/apyrimidinic endonuclease 1. In this study, we describe the cloning of a fragment specifying the chloramphenicol-resistance gene into a SacI vector site. To further test this method, three segments of the lacZ gene were amplified by PCR, and after treatment with the DNA-modifying enzymes, the properly oriented segments were ligated into a SacI-cleaved plasmid. Using the methods described, we were able to assemble PCR products into appropriate structures.

Carbon-Oxygen Lyases↗
PubMed1987

Lability of DNA polymerase alpha correlated with decreased DNA synthesis and increased age in human cells.

DNA excision repair and mitogen-initiated blastogenesis in human cells declined in efficiency as an apparent function of decreased DNA polymerase alpha specific activity with increased age of the cell donor. DNA polymerase alpha isolated from fetal cells contained a single, high-specific-activity enzyme form that could not be further activated and that was stable with regard to enzyme activity and affinity for DNA template-primer. DNA polymerase alpha isolated from adult-derived cells contained both low-specific-activity and high-specific-activity forms. The low-activity enzyme form, which showed low affinity of binding to DNA template-primer, was activated by treatment with phosphatidylinositol, 32P-ATP, and phosphatidylinositol kinase, resulting in a 32P-labeled enzyme that exhibited high affinity of binding to DNA template-primer. The activated enzyme was unstable, exhibiting a loss of 32P-label correlated with the loss of both specific activity and high affinity of binding to DNA template-primer. The data suggest that DNA polymerase alpha isolated from adult-derived human cells has low-activity and high-activity forms. Decreased specific activity of DNA polymerase alpha correlated with increased age of the donor appears to be a function of loss of an enzyme activator molecule resulting in diminished ability of the enzyme to bind DNA template-primer.

Adult↗
PubMed1990

Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40.

Agents discriminating between DNA polymerase alpha and DNA polymerases of class delta (polymerase delta or epsilon) were used to characterize steps in the synthesis of the lagging DNA strand of simian virus 40 during DNA replication in isolated nuclei. The synthesis of lagging-strand intermediates below 40 nucleotides, termed DNA primers (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988), was selectively inhibited by butylphenyl dGTP or by neutralizing DNA polymerase alpha monoclonal antibodies. The synthesis of longer lagging chains of up to 250 nucleotides (Okazaki pieces) was affected to a lesser extent, possibly indirectly, by these agents. Aphidicolin, which inhibits both alpha- and delta-class enzymes, elicited the opposite pattern: DNA primers accumulated in its presence and were not converted into Okazaki pieces. These and previous data suggest that DNA polymerase alpha primase synthesizes DNA primers, whereas another DNA polymerase, presumably DNA polymerase delta or epsilon, mediates the conversion of DNA primers into Okazaki pieces.

Animals↗
PubMed1997

Structural analyses of gp45 sliding clamp interactions during assembly of the bacteriophage T4 DNA polymerase holoenzyme. III. The Gp43 DNA polymerase binds to the same face of the sliding clamp as the clamp loader.

In the preceding paper (Latham, G. J., Bacheller, D. J., Pietroni, P. , and von Hippel, P. H. (1997) J. Biol. Chem. 272, 31677-31684), we demonstrated that the T4 gp44/62-ATP clamp loader binds to the C-terminal face of the gp45 sliding clamp. Here we extend these results by exploring the structural relationship between the gp43 polymerase and the gp45 sliding clamp. Using fluorescence intensity and polarization techniques, as well as photo-cross-linking methods, we present evidence that gp43, like gp44/62, binds to the C-terminal face of gp45. In addition, we show that g43 binds to the gp45 clamp in two distinct interaction modes, depending on the presence or absence of template-primer DNA. When template-primer DNA is present, gp43 binds tightly to gp45 to form the highly processive DNA polymerase holoenzyme. Gp43 also binds to gp45 in the absence of template-primer DNA, but this interaction is more than 100 times weaker than gp43-gp45 binding on DNA. Specific interactions between gp43 and the C-terminal face of gp45 are maintained in both modes of binding. These results underscore the pivotal role of template-primer DNA in modulating the strength of protein-protein interactions during DNA synthesis and provide additional insight into the structural requirements of the replication process.

Adenosine Triphosphate↗
PubMed1994

A new simplified method of gene typing.

SSP-PCR (sequence-specific primer) DNA typing was performed in Terasaki trays using 1.5 microliters of DNA, and the ethidium-stained PCR product was measured by direct fluorometric reading. Elimination of the gel electrophoresis step greatly simplified the SSP method. 17 serological DR specificities were discriminated for 239 DNA samples utilizing the new method, standard SSP, sequence-specific oligonucleotide probe (SSOP), and restriction fragment length polymorphism (PCR-RFLP). Results showed 98% concordance between the SSP-PCR assay and conventional methods. DRB1 alleles were determined by PCR-RFLP in 59 samples, by SSP in 110 samples, and by consensus (all methods) in the remaining samples.

Base Sequence↗
PubMed1984

Interaction of herpes simplex virus-induced DNA polymerase with 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate.

The triphosphate of 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG) competitively inhibits incorporation of dGTP into DNA catalyzed by DNA polymerases specified by both type 1 and type 2 herpes simplex virus. K1 values were estimated to be 33 nM for type 1 and 46 nM for type 2-specified DNA polymerase. DHPG acted as an alternate substrate to dGTP for the virus-specified DNA polymerase. Incorporation of DHPG into DNA resulted in the slowing down of the rate of DNA synthesis. The position of DHPG incorporation was analyzed, and it was found to enter both internal and terminal linkages. DNA which contained DHPG at termini was found to competitively inhibit utilization of activated DNA as primer. DNA polymerase alpha and DNA polymerases from several phosphonoformic acid-resistant herpes simplex virus type 1 strains were examined for sensitivity to 9-(1,3-dihydroxy-2-propoxymethyl)guanine triphosphate. A lack of correlation between the in vivo sensitivities of the virus mutants and the K1 values of the DNA polymerases was noted.

Animals↗
PubMed2001

A random amplified polymorphic DNA (RAPD) primer to assist the identification of a selected strain, aizu K-111 of Panax ginseng and the sequence amplified.

Panax ginseng is widely used as a Chinese medicine, but it takes a long time to reach harvest and to establish its qualified strains. In the course of searching high quality Panax ginseng, we found a useful random amplified polymorphic DNA (RAPD) primer, which showed a 725 base pair band for a selected elite strain Aizu K-111 (now called Kaishusan) including its cultured tissues, while the other strains did not necessarily show this band. We sequenced the DNA fragment amplified and designed primers to improve electrophoretic profiles, based on the sequence.

Base Sequence↗
PubMed1996

Non specific PCR products using rat-derived Pneumocystis carinii dihydrofolate reductase gene-specific primers in DNA amplification of human respiratory samples.

DNA amplification using dihydrofolate reductase (DHFR) primers in bronchoalveolar lavage fluids (BALFs) from patients with Pneumocystis carinii (PC) pneumonia yielded low sensitivity and specificity. Amplified products of BALFs from an AIDS patient without PC pneumonia and five patients with PC pneumonia were cloned and sequenced. All samples showed the same sequence without any homology with DHFR cDNA of rat PC, or with any DHFR sequences in databases at the DNA or amino acid level. The data demonstrate that these DHFR primers amplify a non-specific region of DNA with a sequence unrelated to the human PC DHFR gene both in PC positive and in PC negative samples. This finding precludes the use of these DHFR primers for the diagnosis of PC pneumonia in respiratory specimens.

Base Sequence↗
PubMed1995

3-Nitropyrrole and 5-nitroindole as universal bases in primers for DNA sequencing and PCR.

3-Nitropyrrole and 5-nitroindole have been assessed as universal bases in primers for dideoxy DNA sequencing and in the polymerase chain reaction (PCR). In contrast to a previous report, we have found that the introduction of more than one 3-nitropyrrole residue at dispersed positions into primers significantly reduced their efficiency in PCR and sequencing reactions. Primers containing 5-nitroindole at multiple dispersed positions were similarly affected; for both bases only a small number of substitutions were tolerated. In PCR experiments neither base, when incorporated into primers in codon third positions, was as effective as hypoxanthine, which was incorporated in six codon third positions in a 20mer oligomer. However, primers containing up to four consecutive 5-nitroindole substitutions performed well in both PCR and sequencing reactions. Consecutive 3-nitropyrrole substitutions were tolerated, but less well in comparable reactions.

Base Sequence↗
PubMed1998

Cyclin-dependent kinase inhibitor p21 modulates the DNA primer-template recognition complex.

The p21 protein, a cyclin-dependent kinase (CDK) inhibitor, is capable of binding to both cyclin-CDK and the proliferating cell nuclear antigen (PCNA). Through its binding to PCNA, p21 can regulate the function of PCNA differentially in replication and repair. To gain an understanding of the precise mechanism by which p21 affects PCNA function, we have designed a new assay for replication factor C (RFC)-catalyzed loading of PCNA onto DNA, a method that utilizes a primer-template DNA attached to agarose beads via biotin-streptavidin. Using this assay, we showed that RFC remains transiently associated with PCNA on the DNA after the loading reaction. Addition of p21 did not inhibit RFC-dependent PCNA loading; rather, p21 formed a stable complex with PCNA on the DNA. In contrast, the formation of a p21-PCNA complex on the DNA resulted in the displacement of RFC from the DNA. The nonhydrolyzable analogs of ATP, adenosine-5'-O-(3-thiotriphosphate) (ATPgammaS) and adenyl-imidodiphosphate, each stabilized the primer recognition complex containing RFC and PCNA in the absence of p21. RFC in the ATPgammaS-activated complex was no longer displaced from the DNA by p21. We propose that p21 stimulates the dissociation of the RFC from the PCNA-DNA complex in a process that requires ATP hydrolysis and then inhibits subsequent PCNA-dependent events in DNA replication. The data suggest that the conformation of RFC in the primer recognition complex might change on hydrolysis of ATP. We also suggest that the p21-PCNA complex that remains attached to DNA might function to tether cyclin-CDK complexes to specific regions of the genome.

Adenosine Triphosphate↗
PubMed1992

Fluorescence chemistries for automated primer-directed DNA sequencing.

A major limitation in the applicability of automated DNA sequencing instruments has been the difficulty in using user-defined oligonucleotide primers which allow sequencing reactions to start at any specific point in a region of interest. Recently, new chemistries have become available for fluorescent labeling which will begin to facilitate the use of any oligonucleotide primer with automated DNA sequencers. In this report, we describe several methods for automated primer-directed DNA sequencing, and compare and discuss the relative merits and limitations of these methods.

Automation↗
PubMed1996

The algorithm of estimation of the Km values for primers in DNA synthesis catalyzed by human DNA polymerase alpha.

DNA synthesis with various deoxyribo homo- and heterooligoprimers in the presence of complementary templates was investigated. The lengthening of d(pN)n, primers (n = 1-10) by a unit resulted in an increase of the primer affinity and a maximal rate of polymerization. The coefficient of the affinity enhancement of primers due to formation of one hydrogen bond between primer and template was found to be 1.35. The dependence of the primer affinity and polymerization rate on template-primer structure in solution was analyzed and the objective laws of the changes of the KM and Vmax values were revealed.

Algorithms↗
PubMed1996

Chlorodeoxyadenosine and arabinosylcytosine in patients with acute myelogenous leukemia: pharmacokinetic, pharmacodynamic, and molecular interactions.

The effectiveness of arabinosylcytosine (ara-C) for the treatment of acute myelogenous leukemia (AML) depends on the formation of its active metabolite, the triphosphate of ara-C (ara-CTP). Using biochemical modulation strategies to increase the accumulation of ara-CTP in leukemia blasts, a clinical protocol was designed combining 2-chlorodeoxyadenosine (CdA), an inhibitor of ribonucleotide reductase, and ara-C for adults with AML. The protocol stipulated an infusion of 1 g/m2 of ara-C over 2 hours on day 1. A continuous infusion of CdA (12 mg/m2/d) begun 24 hours later and continued for 5 days. Identical doses of ara-C were administered on days 3, 4, 5, and 6. Pharmacokinetic and pharmacodynamic interactions between CdA and ara-C during therapy were investigated. To complement these studies, molecular actions of the triphosphate of ara-C and CdA on DNA extension by human DNA polymerase alpha in an in vitro model system was conducted. In the circulating leukemia blasts of 7 of the 9 patients studied, ara-CTP pharmacokinetics showed a median 40% increase in the rate of ara-CTP accumulation after 24 hours of CdA infusion. The ex vivo effect of CdA on accumulation of ara-CTP in AML blasts was similar to that during therapy except that the enhancement was less. The DNA synthetic capacity of the circulating blasts was inhibited to a greater extent by administration of CdA and ara-C in combination than by either one alone. Additionally the lowered level of DNA synthesis was maintained until the next infusion of ara-C. Endogenous levels of deoxynucleotides increased 24 hours after ara-C infusion. Administration of CdA in general lowered the concentrations of all dNTPs. DNA pol alpha incorporated CdATP and ara-CTP with high affinity in a DNA primer extending over an oligonucleotide template of defined sequence. Human DNA polymerase alpha extended DNA primers terminated by CdA monophosphate (CdAMP) at its 3'-end by incorporating ara-C monophosphate (ara-CMP). The tandem incorporation of CdAMP and ara-CMP resulted in nearly complete inhibition of DNA primer extension. The insertion of two analogs in sequence, inhibition of ribonucleotide reductase, and the metabolic potentiation of ara-CTP by CdA infusion may be responsible for sustained inhibition of DNA synthesis in the circulating leukemia blasts during therapy with this combination regimen.

Adenosine Triphosphate↗