Large Fragment of DNA Polymerase I from Geobacillus sp. 777: Cloning and Comparison with DNA Polymerases I in Practical Applications.

A truncated gene of DNA polymerase I from the thermophilic bacteria Geobacillus sp. 777 encoding a large fragment of enzyme (LF Gss pol) was cloned and sequenced. The resulting sequence is 1776-bp long and encodes a 592 aa protein with a predicted molecular mass of 69.8 kDa. Enzyme was overexpressed in E. coli, purified by metal-chelate chromatography, and biochemically characterized. The specific activity of LF Gss pol is 104,000 U/mg (one unit of enzyme was defined as the amount of enzyme that incorporated 10 nmol of dNTP into acid insoluble material in 30 min at 65 °C). The properties of LF Gss pol were compared to commercially available large fragments of DNA polymerase I from G. stearothermophilus (LF Bst pol) and Bacillus smithii (LF Bsm pol). Studied enzymes showed maximum activity at similar pH and concentrations of monovalent/divalent ions, whereas LF Gss pol and LF Bst pol were more thermostable than LF Bsm pol. LF Gss pol is more resistant to enzyme inhibitors (SYBR Green I, heparin, ethanol, urea, blood plasma) in comparison with LF Bst pol and LF Bsm pol. LF Gss pol is also suitable for loop-mediated isothermal amplification and whole genome amplification of human genomic DNA.

Effect and mechanism of beta-L-D4A on DNA polymerase alpha.

AIM: To investigate the safety of beta-L-D4A on DNA polymerase alpha. METHODS: Ion exchange chromatography was used to separate DNA polymerase alpha from crude extract of human Hela cells. Detailed kinetic parameters were determined for beta-L-D4A against DNA polymerase alpha. RESULTS: DNA polymerase alpha was purified with 4% yield and 31000 units/mg specific activity. The Michaelis constant (Km = 3.22 micromol/L), 50% inhibition concentration (IC50 = 178.49 micromol/L) and inhibition constant (Ki = 126 micromol/L) of beta-L-D4A were determined by kinetic analysis. CONCLUSION: beta-L-D4A is a more safe nucleoside for hepatitis B virus infection with a lower host toxicity.

Apoptosis of human breast carcinoma cells in the presence of cis-platin and L-/D-PPMP: IV. Modulation of replication complexes and glycolipid: Glycosyltransferases.

Apoptosis of human breast carcinoma cells (SKBR-3, MCF-7, and MDA-468) has been observed after treatment of these cells with anti-cancer drug cis-platin and glycosphingolipid biosynthesis inhibitor L- and D-PPMP, respectively. These drugs initiated apoptosis in a dose-dependent manner as measured by phenotypic morphological changes, by binding of a fluorescent phophatidyl serine-specific dye (PSS-380) onto the outer leaflet of the cell membranes, and by activation of caspases, -3, -8, and -9. It was observed that in two hours very little apoptotic process had started but predominant biochemical changes occurred after 6 h. DNA degradation started after 24 hours of drug treatment. However, very little is known about the stability of the ';Replication Complexes'' during the apoptotic process. DNA helicases are motor proteins that catalyze the melting of genomic DNA during its replication, repair, and recombination processes. Previously, DNA helicase-III was characterized as a component of the replication complexes isolated from embryonic chicken brains as well as breast and colon carcinoma cells. Helicase activities were measured by a novel method (ROME assay), and DNA polymerase-alpha activities were determined by regular chain extension of the nicked ACT-DNA, by determining values obtained from +/- aphidicolin-treated incubation mixtures. In all three breast carcinoma cell lines, a common trend was observed: a decrease of activities of DNA polymerase-alpha and Helicase III. A sharp decrease of activities of the glycolipid sialyltransferases: SAT-2 (CMP-NeuAc; GD3 alpha2-8 sialyltransferase) and SAT-4 (CMP-NeuAc: GM1a alpha2-3 sialyltransferase) was observed in the apoptotic carcinoma cells treated with L-PPMP compared with cis-platin.

Studies on fertilization in the teleost IV. Effects of aphidicolin and camptothecin on chromosome formation in fertilized medaka eggs.

To clarify the mechanisms of fish fertilization, the effects of inhibitors of DNA polymerase-alpha and DNA topoisomerases on nuclear behavior before and after fertilization were examined in eggs of the medaka, Oryzias latipes. Eggs underwent the fertilization process from sperm penetration to karyogamy of pronuclei, even when inseminated and incubated in the continuous presence of aphidicolin (DNA polymerase alpha inhibitor), camptothecin (DNA topoisomerase I inhibitor), etoposide, or beta-lapachone (DNA topoisomerase II inhibitor). However, continuous treatment with aphidicolin or camptothecin during fertilization inhibited the formation of sister chromosomes that were normally separated into blastomeres at the time of the subsequent cleavage. Sister chromosome formation appeared concomitantly with an increase in histone H1 kinase activity at the end of DNA synthesis, 30 min post insemination. However, non-activated eggs that were inseminated in saline containing anesthetic MS222 and aphidicolin had high levels of histone H1 kinase and MAP kinase activities, and transformation of the penetrated sperm nucleus to metaphase chromosomes occurred even in the presence of aphidicolin or camptothecin. The male chromosomes were normally separated into two anaphase chromosome masses upon egg activation. These results suggest that DNA polymerase alpha or DNA topoisomerase I, but not DNA topoisomerase II, may be required for the process by which the mitotic interphase nucleus transforms to separable metaphase chromosomes while the activity of MAP kinase is low, unlike the situation in meiotic division, during which MAP kinase activity is high and DNA replication is not required.

Three-dimensional electron microscopy of the clamp loader small subunit from Pyrococcus furiosus.

An archaeal clamp loader, replication factor C (RFC), consists of two proteins, the small subunit (RFCS) and large subunit (RFCL), whose sequences are both highly homologous to those of the eukaryotic RFC components. We have investigated the oligomeric structure of RFCS from Pyrococcus furiosus by electron microscopy using single-particle analysis. RFCS forms mostly ring-shaped hexamers at pH 9.0, although it tends to form C-shaped tetramers or pentamers at a lower pH (pH 5.5). The three-dimensional (3D) structure of the RFCS hexamer was obtained by random conical tilt reconstruction at 24.0-A resolution. RFCS forms a hexameric ring with outer and inner diameters of 117 and 27 A, respectively, and with a height of about 55 A. The six subunits are arranged in a twisted manner with a sixfold symmetry around the channel. The 3D map revealed that the six subunits are arranged in a head-to-tail configuration. Although the RFC complex consists of RFCS and RFCL in vivo, RFCS alone, together with PCNA, substantially enhanced the DNA synthesizing activity of P. furiosus DNA polymerase I in vitro. The 3D reconstruction of RFCS with catalytic activity provides important insights into the organization mechanism and the functional state of the RFC complex.

Role of fission yeast primase catalytic subunit in the replication checkpoint.

To investigate the cell cycle checkpoint response to aberrant S phase-initiation, we analyzed mutations of the two DNA primase subunit genes of Schizosaccharomyces pombe, spp1(+) and spp2(+) (S. pombe primase 1 and 2). spp1(+) encodes the catalytic subunit that synthesizes the RNA primer, which is then utilized by Polalpha to synthesize the initiation DNA. Here, we reported the isolation of the fission yeast spp1(+) gene and cDNA and the characterization of Spp1 protein and its cellular localization during the cell cycle. Spp1 is essential for cell viability, and thermosensitive mutants of spp1(+) exhibit an allele-specific abnormal mitotic phenotype. Mutations of spp1(+) reduce the steady-state cellular levels of Spp1 protein and compromised the formation of Polalpha-primase complex. The spp1 mutant displaying an aberrant mitotic phenotype also fails to properly activate the Chk1 checkpoint kinase, but not the Cds1 checkpoint kinase. Mutational analysis of Polalpha has previously shown that activation of the replication checkpoint requires the initiation of DNA synthesis by Polalpha. Together, these have led us to propose that suboptimal cellular levels of polalpha-primase complex due to the allele-specific mutations of Spp1 might not allow Polalpha to synthesize initiation DNA efficiently, resulting in failure to activate a checkpoint response. Thus, a functional Spp1 is required for the Chk1-mediated, but not the Cds1-mediated, checkpoint response after an aberrant initiation of DNA synthesis.

Low temperature cycled PCR protocol for Klenow fragment of DNA polymerase I in the presence of proline.

A method for performing cycled PCR at low temperatures, using the thermolabile Klenow fragment of DNA polymerase I, is reported. Application of proline as a buffer additive in the range of 3.0-5.5 M remarkably increases the thermal stability of the polymerase and decreases the denaturation temperature of DNAtemplate. This method might be applicable to a broad spectrum of thermolabile DNA polymerases in cycled PCR and other methods of DNA amplification.

Klenow fragment and DNA polymerase alpha-primase fromserva calf thymus in water-in-oil microemulsions.

The activity of DNA polymerase alpha-primase complex from calf thymus and Klenow fragment of E. coli DNA polymerase 1 has been studied in reverse microemulsions formed by sodium bis(2-ethylhexyl) sulfosuccinate (AOT), sodium dodecylsulfate (SDS), cetyl trimethyl ammonium bromide (CTAB), polyoxyethylene 20 cetyl ether (Brij 58), and Triton X-114 in decane. DNA polymerases were not active in AOT, CTAB, and SDS reverse microemulsions, but these enzymes catalyzed DNA synthesis in Brij 58 and its mixture with other surfactants. We have also found the system composed from the Triton X-114, SDS, CTAB, and Brij 58 (concentration of 128, 25, 15, and 10 mM, respectively) in hexanol-decane (1:12 v/v), in which DNA polymerases revealed maximum activity. The above system was optically transparent, fluid, and stable during a few hours with a water-surfactants molar ratio up to 160. The pH dependence of DNA polymerase activity was not significantly different in comparison with water; however, DNA polymerase was sensitive to ionic strength in microemulsions. The dependence of DNA polymerase activity on w0 was the curve with a few optima. DNA polymerases synthesized more products in water than in reverse microemulsions, and the processivity of Klenow fragment decreased. An increase of the water content resulted in an increase of DNA polymerase processivity.

Engagement of DNA polymerases during apoptosis.

DNA replicative and repair machinery was investigated by means of different techniques, including in vitro nuclear enzymatic assays, immunoelectron microscopy and confocal microscopy, in apoptotic cell lines such as HL-60 treated with methotrexate, P815 and K562 exposed to low temperatures and Friend cells exposed to ionizing radiation. The results showed a shift of DNA polymerase alpha and beta activities. DNA polymerase alpha, which in controls was found to be the principal replicative enzyme driving DNA synthesis, underwent, upon apoptosis, a large decrease of its activity being replaced by DNA polymerase beta which is believed to be associated with DNA repair. Such a modulation was concomitant with a topographical redistribution of both DNA polymerase alpha and the incorporation of BrdUrd throughout the nucleus. Taken together, these results indicate the occurrence of a dramatic response of the DNA machinery, through a possible common or at least similar behaviour when different cell lines are triggered to apoptosis. Although this possibility requires further investigation, these findings suggest an extreme attempt of the cell undergoing apoptosis to preserve its nuclear environment by switching on a repair/defence mechanism during fragmentation and chromatin margination.

[Effect of HH07A, A derivative of hainanensine, on DNA polymerase I].

The effect of HH07A on DNA polymerase I in a cell free system was studied. The results showed that HH07A strongly inhibited the DNA synthesis catalyzed by DNA polymerase I in a dose dependent manner. Further results indicated that the DNA synthesis decreased significantly after DNA polymerase I was preincubated with HH07A. However, no change was found when DNA template was preincubated with HH07A. These results suggest that the inhibitory action of HH07A appeared to be on the enzyme molecules rather than on the DNA template.

Modulation by dietary copper of aflatoxin B1-induced activity of DNA repair enzymes poly (ADP-ribose) polymerase, DNA polymerase beta and DNA ligase.

The activity of some nuclear enzymes associated with DNA repair was examined following aflatoxin B1 administration in rats maintained on different levels of dietary copper. Induction of poly(ADP-ribose) polymerase, DNA polymerase beta and DNA ligase was found to be significantly higher in copper-deficient rats. Copper supplementation, even at marginal doses, was able to bring down the induction to the level observed in normal rats. The results emphasize the protective role of copper against the DNA damaging effects of aflatoxin B1.

Inactivation of human immunodeficiency virus type 1 reverse transcriptase by oltipraz: evidence for the formation of a stable adduct.

Oltipraz (5-pyrazinyl-4-methyl-1,2-dithiole-3-thione), which is undergoing clinical evaluation as an anticarcinogen, also inhibits HIV-1 replication (IC50 approximately equal to 10 microM). The inactivation of RT appears to be a relevant antiviral mechanism since oltipraz blocks viral replication in acutely infected T-cell lines, but is ineffective in chronically infected ACH-2 cells (H. J. Prochaska, W. G. Bornmann, P. Baron, and B. Polsky (1995) Mol. Pharmacol. 48, 15-20). Since a nucleophilic amino acid is a likely target for oltipraz, we assessed whether the conserved cysteine residues of HIV-1 RT (38Cys or 280Cys) were the target(s) for oltipraz, and we synthesized [Me 14C]oltipraz to determine if oltipraz forms a stable adduct with RT. Thus, HIV-2 RT as well as wild-type, 38Cys-->Ser, 280Cys-->Ser, and the Cys-->Ser double mutant of HIV-1 RT were purified from the lysates of transformed Escherichia coli strain DH5 alpha (A. Hizi, M. Shaharabany, R. Tal, and S. H. Hughes (1992) J. Biol. Chem. 267, 1293-1297) via a purification procedure that included (NH4)2SO4 fractionation followed by gel filtration, dye-ligand, and ion-exchange chromatographies. Procion yellow H4R was chosen as the dye-ligand chromatography since it was the most potent and selective inhibitor of RT among seventy reactive dyes that were screened. Mono Q anion-exchange chromatography with diethanolamine (pH 9) resulted in the generation of heterodimeric RT from a predominantly homodimeric enzyme preparation. Because the instability of dilute RT preparations at room temperature rendered the kinetic evaluation of inactivation difficult, we sought to identify conditions that prevent denaturation of these enzymes. High concentrations (25 mM) of MgCl2 had a stabilizing effect. Oltipraz behaved kinetically as an irreversible inhibitor of all RTs purified, and the kinetic constants for the inactivation of these enzymes were not significantly different from wild-type HIV-1 RT (Ki = 17.0 +/- 4.1 microM; k3 = 0.214 +/- 0.051 h-1). In stark contrast, oltipraz neither inhibited nor inactivated the Klenow fragment of DNA polymerase I, whose subdomain structure is similar to the p66 subunit of RT. Wild-type RT was incubated with 60 microM [Me 14C]oltipraz for 4 h and was then subjected to gel filtration chromatography. The [14C] label comigrated with RT with a stoichiometry of binding of 0.88 +/- 0.05 oltipraz per inactivated RT subunit (N = 3 experiments), and the [14C] label remained bound after treatment with 4 M urea.(ABSTRACT TRUNCATED AT 400 WORDS)

Mechanism of caffeine modulation of the antitumor activity of adriamycin.

We examined the effects of a combination of adriamycin (ADR) and caffeine on DNA and protein biosynthesis and on the activities of DNA polymerase alpha and beta in normal and tumor tissue. The decrease in DNA and protein biosynthesis in tumor produced by caffeine combined with ADR were 2.5 and 2.4 times greater, respectively, compared with ADR alone. The combination of caffeine and ADR enhanced the decrease in DNA polymerases activities in the tumor which was induced by ADR, the decreases being 1.8 and 1.6 times greater, respectively, than that of ADR alone. In contrast, these ADR-induced changes in normal tissues were not enhanced by the combination with caffeine. The combination with caffeine had no effect on ADR concentration in normal tissues, but in the tumor, it increased the ADR concentration to 2.1 times that of ADR alone. In vitro, ADR efflux from Ehrlich ascites carcinoma cells was significantly inhibited by exposure to caffeine. These findings indicate that the effect of caffeine on ADR concentration in the cell plays an important role in the mechanism by which caffeine enhances ADR antitumor activity.

Effects of chromium on DNA replication in vitro.

Chromium is an environmentally significant human carcinogen with complicated metabolism and an unknown mechanism of mutagenesis. Chromium(VI) is taken up by cells as the chromate anion and is reduced intracellularly via reactive intermediates to stable Cr(III) species. Chromium(III) forms tight complexes with biological ligands, such as DNA and proteins, which are slow to exchange. In vitro, CrCl3.6H2O primarily interacts with DNA to form outer shell charge complexes with the DNA phosphates. However, at micromolar concentrations, the Cr(III) binds to a low number of saturable tight binding sites on single-stranded M13 DNA. Additional chromium interacts in a nonspecific manner with the DNA and can form intermolecular DNA cross-links. Although high concentrations of Cr(III) inhibit DNA replication, micromolar concentrations of Cr(III) can substitute for Mg2+, weakly activate the Klenow fragment of E. coli DNA polymerase I (Pol I-KF), and act as an enhancer of nucleotide incorporation. Alterations in enzyme kinetics induced by Cr(III) increase DNA polymerase processivity and the rate of polymerase bypass of DNA lesions. This results in an increased rate of spontaneous mutagenesis during DNA replication both in vitro and in vivo. Our results indicate that chromium(III) may contribute to chromate-induced mutagenesis and may be a factor in the initiation of chromium carcinogenesis.

[Preliminary mechanism studies of siwenmycin on the inhibition of nucleic acid syntheses].

Siwenmycin, isolated from a streptomyces culture, is a new member of aclacinomycin analogues. It exhibited a remarkable inhibitory effect on the biosynthesis of DNA and RNA in vivo. In order to recognize the mechanism of the inhibitions, the reactions of siwenmycin to DNA template, DNA polymerase I and RNA polymerase were studied. Experimental results showed that siwenmycin could intercalate DNA, but it did not inhibit DNA polymerase I-mediated DNA repair replication and T7RNA polymerase-mediated DNA transcription. This indicates that siwenmycin is not a damage to the DNA template function, nor will it inhibit DNA polymerase I and T7RNA polymerase, though it can intercalate DNA.

Mechanism of inhibition of DNA synthesis by 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl)uracil.

The mechanism of the inhibitory action of 1-beta-D-arabinofuranosyl-E-5-(2-bromovinyl) uracil triphosphate (BV-araUTP) on DNA synthesis by Escherichia coli DNA polymerase I Klenow fragment was studied. Acting as a chain terminator, BV-araUTP inhibited DNA synthesis by Klenow fragment more effectively than 2',3'-dideoxythymidine triphosphate (ddTTP). However, the incorporation sites of BV-araU monophosphate were restricted at consecutive dTMP sequence whereas ddTMP was incorporated at every dTMP site.

Partial purification and characterization of a beta-like DNA polymerase from Leishmania mexicana.

Deoxyribonucleic acid polymerase beta (EC 2.7.7.7) from the lower eukaryotic parasitic protozoan Leishmania mexicana has been partially purified over 9,000 fold and characterized for the very first time. Like mammalian DNA polymerase beta the protozoan enzyme is of low molecular weight (40,000), has a broad pH range, and is resistant to inhibition by N-ethylmaleimide and aphidicolin. It is unlike mammalian DNA polymerase beta in utilization of various templates and response to various inhibitors and sensitivity to high ionic strength, but similar to a beta-like enzyme from a related organism Crithidia fasciculata. It is estimated that this enzyme constitutes 20% of the polymerase activity of the crude cell extract.

Interaction of DNA with the Klenow fragment of DNA polymerase I studied by time-resolved fluorescence spectroscopy.

The interaction of a fluorescent duplex DNA oligomer with the Klenow fragment of DNA polymerase I from Escherichia coli has been studied in solution by using time-resolved fluorescence spectroscopy. An aminonaphthalenesulfonate (dansyl) fluorescent probe was linked by a propyl chain to a C5-modified uridine base located at a specific site in the primer strand of the DNA oligomer. The fluorescent oligomer bound tightly to the Klenow fragment (KD = 7.9 nM), and the probe's position within the DNA-protein complex was varied by stepwise elongation of the primer strand upon addition of the appropriate deoxynucleoside triphosphates. The decay of the total fluorescence intensity and the polarization anisotropy were measured with a picosecond laser and a time-correlated single photon counting system. The fluorescence lifetimes, the correlation time for internal rotation, and the angular range of internal rotation varied according to the probe's position within the DNA-protein complex. These results showed that five or six bases of the primer strand upstream of the 3' terminus were in contact with the protein and that within this contact region there were differences in the degree of solvent accessibility and the closeness of contact. Further, a minor binding mode of the DNA-protein complex was identified, on the basis of heterogeneity of the probe environment observed when the probe was positioned seven bases upstream from the primer 3' terminus, which resulted in a distinctive "dip and rise" in the anisotropy decay. Experiments with an epoxy-terminated DNA oligomer and a site-directed mutant protein established that the labeled DNA was binding at the polymerase active site (major form) and at the spatially distinct 3'----5' exonuclease active site (minor form). The abundance of each of these distinct binding modes of the DNA-protein complex was estimated under solution conditions by analyzing the anisotropy decay of the dansyl probe. About 12% of the labeled DNA was bound at the 3'----5' exonuclease site. This method should be useful for investigating the editing mechanism of this important enzyme.

Sequence-dependent termination of mammalian DNA polymerase reaction by a new platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato)-2-plati num(II) monohydrate).

We examined the mechanism of the inhibition of DNA synthesis by a new platinum compound, (-)-(R)-2-aminomethylpyrrolidine(1,1-cyclobutane-dicarboxylato+ ++)-2-platinum(II) monohydrate (DWA-2114R), a derivative of the antitumor drug cis-diamminedichloroplatinum(II) (CDDP), using prokaryotic and eukaryotic DNA polymerases. Preincubating activated DNA with CDDP or DWA-2114R reduced its template activity for prokaryotic and eukaryotic DNA polymerases in a dose-dependent manner. DWA2114R required six times greater drug concentration and two times longer incubation time to show the same decrease of the template activity compared to CDDP. Treatment of primed pUC118 ssDNA templates with the two drugs followed by second-strand synthesis by prokaryotic and eukaryotic DNA polymerases revealed that DWA2114R bound to DNA in a similar manner to CDDP and these adducts blocked DNA elongation by DNA polymerases of eukaryotes as well as of prokaryotes. With these two drugs, the elongations by E. coli DNA polymerase I (Klenow fragment), T7 DNA polymerase and calf thymus DNA polymerase alpha were strongly arrested at guanine-guanine sequences (GG). Stop bands were also observed at adenine-guanine sequences (AG) guanine-adenine-guanine sequences (GAG) and mono-guanine sequence (G). Calf testis DNA polymerase beta was also arrested efficiently at AG, GAG and G, but much more weakly at GG. This pattern was common to DWA2114R and CDDP.