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H Maki

Publications and source records attributed to H Maki.

At least 73 records · Page 4Linked to original sources
PubMed1991

A strong mutator effect caused by an amino acid change in the alpha subunit of DNA polymerase III of Escherichia coli.

Most potent mutators heretofore detected in Escherichia coli are associated with defects in epsilon subunit of DNA polymerase III, encoded by the dnaQ gene. To elucidate the role of the alpha subunit, the catalytic subunit of the polymerase, in maintaining the high fidelity of DNA replication, we isolated a mutator mutant, the mutation (dnaE173) of which resides on the dnaE gene, encoding the alpha subunit. The dnaE173 mutant was unable to grow in salt-free L broth at temperatures exceeding 44.5 degrees C and exhibited an increased frequency of spontaneous mutations, 1,000 to 10,000-fold the wild type level, at permissive temperatures. The mutator effect of dnaE173 mutation is dominant over the wild type allele. These phenotypes are caused by a single base substitution, resulting in one amino acid change, Glu612 (GAA)----Lys(AAA), in the alpha subunit molecule. DNA polymerase III purified from the dnaE173 mutant contained both alpha and epsilon subunits, in a normal molar ratio. We found no differences between wild type and mutant polymerases in the Vmax, thermolabilities, and salt sensitivities. However, the apparent Km for the substrate nucleotide of the mutant polymerase was 1/6 of that determined with the wild type polymerase. Although the mutant polymerase retained a normal level of 3'----5' exonuclease activity, the proofreading capacity determined by "turnover assay" was significantly lower in the mutant polymerase, as compared with findings in the normal enzyme. It seems likely that the enhanced mutability in the dnaE173 strain results from, at least in part, a defect in the editing function of DNA polymerase III, and further suggests that a portion of the alpha subunit in which the amino acid change resides may be important for the proper setting of the two subunits at the replication fork so as to facilitate efficient editing during the DNA replication.

Alleles↗
PubMed1991

Polyamines and the Cell Cycle of Catharanthus roseus Cells in Culture.

Investigation was made on the effect of partial depletion of polyamines (PAs), induced by treatment with inhibitors of the biosynthesis of PAs, on the distribution of cells at each phase of the cell cycle in Catharanthus roseus (L.) G. Don. cells in suspension cultures, using flow cytometry. More cells treated with inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC) were accumulated in the G(1) phase than those in the control, while the treatment with an inhibitor of spermidine (SPD) synthase showed no effect on the distribution of cells. The endogenous levels of the PAs, putrescine (PUT), SPD, and spermine (SPM), were determined during the cell cycle in synchronous cultures of C. roseus. Two peaks of endogenous level of PAs, in particular, of PUT and SPD, were observed during the cell cycle. Levels of PAs increased markedly prior to synthesis of DNA in the S phase and prior to cytokinesis. Activities of ADC and ODC were also assayed during the cell cycle. Activities of ADC was much higher than that of ODC throughout the cell cycle, but both activities of ODC and ADC changed in concert with changes in levels of PAs. Therefore, it is suggested that these enzymes may regulate PA levels during the cell cycle. These results indicate that inhibitors of PUT biosynthesis caused the suppression of cell proliferation by prevention of the progression of the cell cycle, probably from the G(1) to the S phase, and PUT may play more important roles in the progression of the cell cycle than other PAs.

Journal Article↗
PubMed1991

Use of universal and type-specific primers in the polymerase chain reaction for the detection and typing of genital human papillomaviruses.

By using polymerase chain reaction (PCR), we have developed a system for type-specific as well as universal detection of genital human papillomaviruses (HPVs). Primers and probes for specific detection of HPV-16, -18 and -33 were synthesized from the E7 open reading frame (ORF). They were capable of detecting corresponding HPV types with high specificity and sensitivity. Primers for detection of a broad spectrum of HPV (universal primers) were synthesized from the L1 ORF. The universal primers were shown to be capable of amplifying HPV-6b, -11, -16, -18, -33, -52b and -58. The system was applied to various cervical tissue specimens from Japanese patients. They consisted of 26 normal specimens, 18 from cervical dysplasias and 29 from cervical carcinomas. HPV was detected in none of the normal specimens. On the other hand, many of the specimens from cervical dysplasias and carcinomas were found to be positive for HPV, especially HPV-16. Except for one, all the specimens which were positive with the type-specific PCRs were also positive with the universal PCR. Furthermore, substantial numbers of specimens were found to be positive only with the universal PCR. Cloning and sequencing of DNA segments amplified by the universal primers were undertaken to characterize some of the unknown HPVs. Our PCR system may thus be useful for the specific detection of the three major types of oncogenic HPVs and also for the detection of a broad spectrum of HPVs including possibly novel HPV types.

Cell Line↗
PubMed1990

Purification, structure, and biochemical properties of human O6-methylguanine-DNA methyltransferase.

The level of O6-methylguanine-DNA methyltransferase activity in a human cell line carrying a 1.1-kilobase cDNA fragment was about 50 times higher than that found in ordinary methyltransferase-proficient (Mer+) cell lines (Hayakawa, H., Koike, G., and Sekiguchi, M. (1990) J. Mol. Biol. 213, 739-747). Taking advantage of this overproduction, the enzyme was purified to apparent physical homogeneity and the physical and biochemical properties investigated. A single polypeptide with a molecular weight of approximately 25,000 was detected on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the most highly purified preparation. The Stokes radius of 22.5 A and the sedimentation coefficient of 2.0 S were obtained, from which the molecular weight of the native form of the enzyme was calculated to be 19,000. After digestion with lysyl endopeptidase, peptide fragments of the protein were isolated and sequenced. The amino acid sequences of these peptides and the amino acid composition of the protein were in good agreement with those deduced from the nucleotide sequence of the cloned cDNA. The purified enzyme catalyzed transfer of methyl groups from O6-methylguanine and O4-methylthymine, but not from methylphosphotriesters, of methylated DNA to the enzyme molecule.

Amino Acid Sequence↗
PubMed1990

Molecular mechanisms of replicational fidelity in Escherichia coli.

DNA polymerase III holoenzyme is responsible for chromosomal DNA synthesis in Escherichia coli and seems to be a major determinant of the fidelity of replication of this organism. Among ten different subunits of the holoenzyme, the alpha subunit, encoded by the dnaE gene, has a polymerase activity, while the epsilon subunit, encoded by the dnaQ gene, is a proofreader with a 3'-5' exonuclease activity. Using poly(dA)/oligo(dT)20 as a template-primer, misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and exonucleolytic editing of those mispairs by the epsilon subunit were investigated. When the polymerization reaction was performed with the alpha subunit, dCMP and dGMP but not dAMP were misincorporated. This would suggest that the polymerase might have a base-selecting function to avoid dA:dA mispairing. A subassembly of the DNA polymerase III consisting of alpha, epsilon, and theta subunits misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit may correct the dC:dA but not the dG:dA mispair. Addition of a protein encoded by the mutT gene, defects of which cause AT to CG transversions in vivo, diminished the misincorporation of dGMP onto poly(dA) template by the alpha subunit. A dGTPase activity was associated with the MutT protein. The significance of the dGTPase activity in the prevention of dG:dA mispairing is discussed.

Bacterial Proteins↗
PubMed1989

A specific role of MutT protein: to prevent dG.dA mispairing in DNA replication.

Occurrence of the transversion mutation A.T to C.G is specifically enhanced in Escherichia coli mutT mutants. With the aid of the cloned mutT gene, the MutT protein, which has a molecular mass of 15 kilodaltons, was overproduced and purified to near homogeneity. The protein catalyzes hydrolysis of dGTP to dGMP. dGDP and GTP were also hydrolyzed by the protein, but at a lower rate than seen with dGTP. No other deoxynucleoside triphosphates were hydrolyzed. Using poly(dA).(dT)20 as a template-primer, we investigated the misincorporation of dGMP, dCMP, and dAMP by the alpha subunit and the core of E. coli DNA polymerase III. When the polymerization reaction was performed with the alpha subunit, both dCMP and dGMP were misincorporated. The core, composed of alpha, epsilon, and theta subunits, misincorporated only dGMP. This would imply that the proofreading function of the epsilon subunit of DNA polymerase III may correct the dC.dA mispair but not the dG.dA mispair. Misincorporation of dAMP was not observed in reactions with the alpha subunit or core. The misincorporation of dGMP, but not dCMP, was almost completely suppressed by adding purified MutT protein to the reaction mixture. Under these conditions, only a portion of dGTP present in the reaction mixture was degraded. It is therefore likely that the MutT protein may prevent dGMP misincorporation by degrading a specific form of dGTP, probably the syn form, which can pair with deoxyadenosine.

Adenine↗
PubMed1989

Lethal effect and potentially lethal damage recovery in cultured mammalian cells irradiated by neutron-capture beams.

Cell killing and potentially lethal damage (PLD) recovery in Hela cells were examined after irradiation in suspension using a 10B neutron-capture beam. Thermal-neutron irradiation in the medium containing from 0.09 to 9 mM boron-10 (0.9-90 micrograms/g 10B, 90 per cent enriched boric acid) resulted in steeper survival curves than in the boron-free medium. The relative biological effectiveness (r.b.e.), expressed as the Do ratio, increased from 2.2 +/- 0.1 for the control without boron-10 to 5.4 +/- 0.3 in the medium containing 0.9 mM boron-10. The r.b.e. value was 5.2 +/- 0.5 at a boron-10 concentration of 9 mM, and did not increase when the boron-10 concentration was increased from 0.9 to 9 mM. It was suggested that the observed plateau of r.b.e. at boron-10 concentrations higher than 0.9 mM was due to the dominancy of the 10B(n, alpha)7Li reaction for dose accumulation. PLD recovery after the neutron irradiation containing 0.9 mM boron-10 was not observed, while a small effect was observed after irradiation in the thermal neutron beam, although much less than after gamma rays. The results show that the neutron-capture beam is an excellent beam for radiotherapy if the boron-10 concentration in tumour cells could be elevated to about 1 mM.

Boron↗
PubMed1989

Suppression of dnaE nonsense mutations by pcbA1.

DNA polymerase III has been recognized as the required replication enzyme in Escherichia coli. The synthesis subunit of DNA polymerase III holoenzyme (alpha subunit) is encoded by the dnaE gene. We have reported that E. coli cells can survive and grow in the absence of a functional dnaE gene product if DNA polymerase I and the pcbA1 mutation are present. Existing mutations in the dnaE gene have been conditionally defective thermolabile mutations. We report here construction of nonsense mutations in the dnaE gene by use of a temperature-sensitive suppressor mutation to permit survival at the permissive temperature (32 degrees C). Introduction of the pcbA1 mutation eliminated the temperature-sensitive phenotype. We confirmed by immunoblotting the lack of detectable alpha subunit at 43 degrees C.

Blotting, Western↗
PubMed1989

Secretion of Mr 46,000 protein from human hepatoma cells treated with tumor promotors is regulated by c-myc gene expression.

Previously an Mr 46,000 protein (named p46) was shown to be induced in the culture medium of human hepatoblastoma cells, Huh-6 Cl-5, treated with 12-O-tetradecanoyl phorbol-13-acetate (TPA). For further characterization of p46, Huh-7 Cl-4, another line of well differentiated liver cells, was incubated in the presence and absence of TPA, and proteins were labeled with [35S]methionine, and the proteins secreted into the medium were analyzed by one- and two-dimensional sodium dodecyl sulfate polyacrylamide gel electrophoresis. A protein of Mr 46,000 was observed in the culture medium of Huh-7 Cl-4 cells treated by TPA or plated at low density, but only slightly in the culture medium of Huh-7 Cl-4 cells plated at high density. As these results suggested that the expression of p46 was regulated by a factor that was activated in the growth state and by TPA treatment, the effect of introduction of the competence gene c-myc into Huh-7 Cl-4 cells was examined. All 37 independent transfected colonies obtained showed enhanced expression of p46. As a control, the c-Ha-ras gene was introduced into Huh-7 Cl-4 cells and shown not to enhance expression of p46. These results strongly suggested that the expression of p46 was regulated by the competence gene c-myc.

Carcinoma, Hepatocellular↗
PubMed1988

DNA Polymerase III holoenzyme of Escherichia coli. IV. The holoenzyme is an asymmetric dimer with twin active sites.

Pol III, a subassembly of Escherichia coli DNA polymerase III holoenzyme lacking only the auxiliary beta subunit, was purified to homogeneity by an improved procedure. This assembly consists of nine different polypeptides, likely in a 1:1 stoichiometry: a catalytic core (pol III) of alpha (132 kDa), epsilon (27 kDa), and theta (10 kDa), and six auxiliary subunits: tau (71 kDa), gamma (52 kDa), delta (35 kDa), delta' (33 kDa), chi (15 kDa), and psi (12 kDa). The assembly behaves on gel filtration as a particle of about 800 kDa, indicating a content of two each of the subunits. A new procedure for purifying the core yielded a novel dimeric form which may provide the foundation for the dimeric nature of the more complex pol III and holoenzyme forms. Pol III readily dissociates into several subassemblies including pol III', likely a dimeric core with two tau subunits. The holoenzyme, purified by a similar procedure with ATP and Mg2+ present throughout, retained the beta subunit (37 kDa) as well as all the subunits present in pol III; the mass of the holoenzyme was estimated to be 900 kDa. The isolated initiation complex of holoenzyme with a primed template DNA and the elongation complex (formed in the presence of three deoxynucleoside triphosphates) had the same composition and stoichiometry as observed for pol III with two beta dimers in addition. An initiation complex assembled from a mixture of monomeric pol III core, gamma 2 delta delta' chi psi complex (gamma complex), beta, and tau retained the core, one beta dimer, and two tau subunits but was deficient in the gamma complex. When tau was omitted from the assembly mixture, the initiation complex contained one or two gamma complexes instead of the tau subunit. Based on these data, pol III holoenzyme is judged to be an asymmetric dimeric particle with twin pol III core active sites and two different sets of auxiliary units designed to achieve essentially concurrent replication of both leading and lagging strand templates.

Binding Sites↗
PubMed1988

Site-directed mutagenesis with Escherichia coli DNA polymerase III holoenzyme.

Escherichia coli DNA polymerase III holoenzyme was used to synthesize double-stranded DNA from M13 single-stranded DNA hybridized to a phosphorylated synthetic oligodeoxynucleotide containing a nucleotide substitution. The resulting DNA was transfected into E. coli JM101 without further treatment. Sequence analysis of randomly chosen phage clones revealed that the efficiency of mutagenesis was nearly 50%, which is the theoretical maximum. Treatment with DNA ligase after DNA synthesis was not necessary to obtain high efficiency of mutagenesis. Thus, use of DNA polymerase III holoenzyme provides a simple and efficient procedure for site-directed mutagenesis.

Bacterial Proteins↗
PubMed1987

Proofreading by DNA polymerase III of Escherichia coli depends on cooperative interaction of the polymerase and exonuclease subunits.

The polymerase subunit (alpha) of Escherichia coli DNA polymerase III holoenzyme and the 3'----5' exonuclease subunit (epsilon) are each less active separately than together in the holoenzyme core (an assembly of alpha, epsilon, and theta subunits). In a complex formed from purified alpha and epsilon subunits, polymerase activity increased 2-fold, and that of the 3'----5' exonuclease increased 10- to 80-fold. The alpha-epsilon complex contains one each of the subunits as does the core. Stimulation of 3'----5' exonuclease activity is due mainly to a greatly increased affinity of the epsilon subunit for the 3'-hydroxyl terminus, resulting from DNA binding by the alpha subunit. Proofreading in the course of DNA synthesis by the alpha-epsilon complex was indistinguishable from that of the core. These findings identify the participation of the alpha subunit in proofreading by polymerase III holoenzyme and suggest that the fidelity of DNA replication may be influenced by the relative levels of the alpha and epsilon subunits in the cell.

Bacterial Proteins↗
PubMed1986

Structure and function of dnaQ and mutD mutators of Escherichia coli.

The nucleotide sequences of the recessive dnaQ49 and the dominant mutD5 mutator were determined. The dnaQ49 mutator has a single base substitution in the dnaQ gene, thus causing one amino acid change, 96Val (GTG)----Gly (GGG), in the DnaQ protein (epsilon subunit of DNA polymerase III holoenzyme). The mutD5 mutator possesses two base substitutions in the same gene, resulting in two amino acid changes, 73Leu (TTG)----Trp (TGG) and 164Ala (GCA)----Val (GTA), which were designated the mutD52 and mutD51 mutations, respectively. Construction of chimaeric genes carrying one or two of these mutations revealed: either mutD51 or mutD52 alone causes the dominant mutator phenotype when present in a multi-copy plasmid; mutator phenotype when present in a low-copy plasmid; the dominant mutD51 mutator activity is suppressed by the dnaQ49 mutation when both mutations are present in the same gene. Based on these findings, we devised a model for the action of these mutators.

Amino Acid Sequence↗
PubMed1986

Cell inactivation and DNA single- and double-strand breaks in cultured mammalian cells irradiated by a thermal neutron beam.

The effects on the cellular viability and induction and repair kinetics of DNA strand breaks in HeLa cells were examined after exposure to a thermal neutron beam and compared with those after gamma-irradiation. The thermal neutron survival curve had no initial shoulder. The relative biological effectiveness (r.b.e.) value of the neutron beam was determined to be 2.2 for cell killing (ratio of D0 values), 1.8 and 0.89 for single strand breakage (ssb) by alkaline sedimentation and alkaline elution respectively, and for double strand breakage (dsb) 2.6 by neutral elution. No difference was observed between thermal neutrons and gamma-rays in the repair kinetics of ssb and dsb. It is suggested that the effect induced by the intracellular nuclear reaction, 14N(n,p)14C is mainly responsible for the high r.b.e. values observed.

Cell Survival↗
PubMed1986

Regulation of nitrate reductase activity in cultured spinach cells as studied by an enzyme-linked immunosorbent assay.

An enzyme-linked immunosorbent assay permitting the determination of nanogram quantities of nitrate reductase (NR) in cultured spinach cells has been developed and used for studies of the mechanism by which NR activity is regulated as a function of culture age. When 8-day old spinach cells were transferred to fresh medium, NR activity increased markedly in 2 days and thereafter decreased gradually until it became undetectable on the 10th day after the transfer. Determination of the amounts of NR by the immunosorbent assay indicated that the unique alteration of NR activity could be accounted for by the concomitant change in the amount of NR protein. Immunoblotting analysis of the subunit of NR also supported this result. It is concluded that the regulation of NR in spinach cells as a function of culture age is mediated by changes in the amount of the enzyme protein rather than by activation and inactivation of the preexisting proteins.

Journal Article↗
PubMed1985

The polymerase subunit of DNA polymerase III of Escherichia coli. I. Amplification of the dnaE gene product and polymerase activity of the alpha subunit.

The Escherichia coli dnaE gene, which encodes the alpha subunit of DNA polymerase III (pol III) holoenzyme, has been cloned in a plasmid containing the PL promoter of phage lambda and thermally induced to overproduce the alpha subunit. In cells carrying this plasmid (pKH167), the alpha subunit was amplified, after heat induction, to a level of about 0.2% of the total cellular protein. Polymerase activity was assayed in three ways: (i) gap-filling by pol III holoenzyme and subassemblies of it, (ii) the extensive replication of a primed, single-stranded DNA circle only by pol III holoenzyme, and (iii) complementation of a crude, inactive pol III holoenzyme (temperature-sensitive dnaE mutant fraction) in replication of a primed, single-stranded DNA circle. Amplification of the alpha subunit raised the polymerase level 10-fold in assay (i), indicative of the dependence of pol III gap-filling activity on this polypeptide; pol III holoenzyme activity remained unaffected (assay (ii)), but the complementation activity was raised 5-fold (assay (iii)). Thus, the elevated alpha subunit (free or in a subassembly form) can substitute in vitro for a defective alpha subunit in pol III holoenzyme, but cannot increase the in vivo level of about eight pol III holoenzyme molecules per cell. This low level of pol III holoenzyme is fixed in wild type cells (bearing no plasmid) despite the presence of a 5-fold excess of the alpha subunit, as inferred from the various assays. These results suggest that the low level of pol III holoenzyme is determined by a factor or factors other than the level of the alpha subunit.

Bacteriophage lambda↗