PubMed Health⌕ Search

SEARCH · PubMed Health

Results for “DNA Polymerase III”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 recordsLinked to original sources
PubMed1976

Mechanism of DNA elongation catalyzed by Escherichia coli DNA polymerase III, dnaZ protein, and DNA elongation factors I and III.

Elongation of a primed single-stranded DNA template catalyzed by E. coli DNA polymerase III (DNA nucleotidyltransferase, deoxynucleosidetriphosphate:DNA deoxynucleotidyltransferase, EC 2.7.7.7) requires dnaZ protein and two other protein factors, DNA elongation factors I and III. The reaction occurs by the following mechanism: (i) dnaZ protein and DNA elongation factor III together catalyze the transfer of DNA elongation factor I to a primed DNA template. This transfer reaction requires ATP or dATP in addition to dnaZ protein, DNA elongation factors I and III, and primed template; it does not require DNA polymerase III. (ii) DNA polymerase III binds to the complex of DNA elongation factor I with primed template; it does not bind to primed template which is not complexed with DNA elongation factor I. This binding reaction proceeds in the absence of ATP or dATP as cofactor, dnaZ protein, and DNA elongation factor III and without additional DNA elongation factor I. (iii) The complex of DNA polymerase III, DNA elongation factor I, and primed template catalyzes DNA synthesis upon the addition of dNTPs.

Adenosine Triphosphate↗
PubMed1978

Relationship of Bacillus subtilis DNA polymerase III to bacteriophage PBS2-induced DNA polymerase and to the replication of uracil-containing DNA.

In vivo studies of PBS2 phage replication in a temperature-sensitive Bacillus subtilis DNA polymerase III (Pol III) mutant and a temperature-resistant revertant of this mutant have suggested the possible involvement of Pol III in PBS2 DNA synthesis. Previous results with 6-(p-hydroxyphenylazo)-uracil (HPUra), a specific inhibitor of Pol III and DNA replication in uninfected cells, suggest that Pol III is not involved in phage DNA replication, due to its resistance to this drug. Experiments were designed to examine possible explanations for this apparent contradiction. First, assays of the host Pol III and the phage-induced DNA polymerase activities in extracts indicated that a labile Pol III did not result in a labile phage-induced enzyme, suggesting that this new polymerase is not a modified HPUra-resistant form of Pol III. Indeed the purified phage-induced enzyme was resistant to the active, reduced form of HPUra under all assay conditions tested. Since in vitro Pol III was capable of replicating the uracil-containing DNA found in this phage, the sensitivity of the purified enzyme to reduced HPUra was examined using phage DNA as template-primer and dUTP as substrate; these new substrates did not affect the sensitivity of the host enzyme to the drug.

Bacillus cereus↗
PubMed1975

On the DNA polymerase III of mouse myeloma: partial purification and characterization.

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...

Animals↗
PubMed1979

DNA polymerase III of Escherichia coli. Purification and identification of subunits.

DNA polymerase III, the core of the DNA polymerase III holoenzyme, has been purified 28,000-fold to 97% homogeneity from Escherichia coli HMS-83. The enzyme contains subunits: alpha, epsilon, and theta of 140,000, 25,000, and 10,000 daltons, respectively. The alpha subunit has been previously shown to be a component of both DNA polymerase III and the more complex DNA polymerase III holoenzyme (Livingston, D.M., Hinkle, D., and Richardson, C. (1975) J. Biol. Chem. 250, 461-469; McHenry, C., and Kornberg, A. (1977) J. Biol. Chem. 252, 6478-6484). It is demonstrated here that the epsilon and theta subunits are also subunits of the DNA polymerase III holoenzyme. Thus, the DNA polymerase III holoenzyme contains at least six different subunits. Our preparation has both the 3' leads to 5' and 5' leads to 3' exonuclease activities previously assigned to DNA polymerase III (Livingston, D., and Richardson, C. (1975) J. Biol. Chem. 250, 470-478).

DNA Polymerase III↗
PubMed1976

Purification and characterization of DNA polymerase III from Bacillus subtilis.

DNA polymerase III from Bacillus subtilis has been purified about 4,500-fold. Disc gel electrophoresis of the purified fraction reveals a single major protein band which co-migrates with the polymerase activity. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the polymerase yields a single, 166,000 dalton band. The hydrodynamic properties of the enzyme are ionic strength-dependent. The average values from determinations in high and low salt are 7.6 S for the sedimentation coefficient and 52 A for the Stokes radius. These two parameters indicate a molecular weight for the native enzyme of 160,000. Therefore, the enzyme appears to contain a single, long, polypeptide chain. The enzyme has no endonuclease activity but does have single strand specific exonuclease activity. Hydrolysis is initiated exclusively from the 3' terminus yielding 5' mononucleotides, and a dinucleotide is the limit of digestion. The exonuclease activity has an ionic strength dependence of pH optimum similar to that of the polymerase but appears to be more fastidious in its divalent metal requirement. The mode of attack by the enzyme is strictly distributive. The activity of the exonuclease decreases markedly with increasing substrate size. Two opposing mechanisms account quantitatively for this effect--intrinsic competitive inhibition by interior substrate nucleotides and increasing accessibility of the substrate terminus to the enzyme with increasing chain length. The polymerase synthesizes DNA in the 5' leads to 3' direction and the apparent Km for each of the deoxyribonucleoside triphosphates is about 1 muM. The polymerase replicates RNA-primed, phiX174 DNA in the presence of Escherichia coli elongation Factors I and II. In contrast to polymerase III, B. subtilis DNA polymerase II has no detectable nuclease activity.

Bacillus subtilis↗
PubMed1979

The delta subunit of Escherichia coli DNA polymerase III holoenzyme is the dnaX gene product.

The delta subunit of DNA polymerase III holoenzyme has been purified extensively with an assay for phi X174 DNA synthesis using core (pol III) and beta and gamma subunits. Either the purified delta subunit or the purified DNA polymerase III holoenzyme can complement a defective enzyme fraction from the conditional replication mutant SG133 described by Sevastopoulos et al. [Sevastopoulas, C.G., Wehr, C.T. & Glaser, D. A. (1977) Proc. Natl. Acad. Sci. USA 74, 3485-3489]. It has been established by Henson et al. [Henson, J.M., Chu, H., Irwin, C.A. & Walker, J.R. (1979) Genetics 92, 1,41-1059] that SG133 has two temperature-sensitive mutations, called dnaX and dnaY. The crude enzyme source from dnaX can be complemented by the delta subunit and by DNA polymerase III holoenzyme. By contrast, the core DNA polymerase III and the beta and gamma subunits are unable to complement this defective enzyme fraction. Thus, the delta subunit of DNA polymerase III holoenzyme appears to be the dnaX gene product of Escherichia coli.

Bacteriophage phi X 174↗
PubMed1977

Bacillus subtilis DNA polymerase III is required for the replication of DNA of bacteriophages SPP-1 and phi 105.

The replication of the Bacillus subtilis bacteriophages SPP-1 and phi 105 is sensitive to 6-(p-hydroxyphenylazo)-uracil (HPUra), a selective inhibitor of replicative DNA synthesis of B. subtilis which acts specifically at the levels of a replication-specific polymerase, DNA polymerase III (pol III). The origin of the HPUra-sensitive polymerase required for phage replication was examined by comparison of the drug sensitivity of phage development in a normosensitive host with that in a host carrying azp-12, a polC mutation that specifies production of an HPUra-resistant pol III. azp-12 specified HPUra-resistant phage host pol III. The host polIII requirement for SPP-1 replication also was confirmed by the demonstration that phage development was temperature sensitive in a host mutant carrying the polC mutation mut-1 (ts). Examination of the pol III activity of crude and purified cell-free preparations derived from phage-infected cells did not indicate any detectable changes in the specific activity, purification behavior, or drug sensitivity of the enzyme.

Bacillus subtilis↗
PubMed1979

Antibody to B. subtilis DNA polymerase III: use in enzyme purification and examination of homology among replication-specific DNA polymerases.

Bacillus subtilis DNA polymerase III (pol III), an arylhydrazinopyrimidine-sensitive, replication-specific enzyme, was used to generate a non-precipitating rabbit antibody which specifically inhibited pol III activity in vitro. The antibody was used to examine structural relationships among several DNA polymerases, and it was linked covalently to agarose; the antibody:agarose was employed to develop a rapid, selective method of purification of catalytically active B. subtilis pol III.

Animals↗
PubMed1977

DNA polymerase III holoenzyme of Escherichia coli. Purification and resolution into subunits.

DNA polymerase III holoenzyme has been purified from Escherichia coli HMS-83, using, as an assay, the conversion of coliphage G4 single-stranded DNA to the duplex replicative form. The holoenzyme consists of at least four different subunits: alpha, beta, gamma, and delta of 140,000, 40,000, 52,000, and 32,000 daltons, respectively. The alpha subunit is DNA polymerase III, the dnaE gene product. The holoenzyme has been resolved by phosphocellulose chromatography into an alpha - gamma - delta complex and a subunit beta (copolymerase III*); neither possesses detectable activity in the G4 system but together reconstitute holoenzyme-like activity. The alpha - gamma - delta complex has been further resolved to yield a gamma - delta complex which reconstitutes alpha - gamma - delta activity when added to DNA polymerase III. The gamma - delta complex contains a product of the dnaZ gene and has been purified from a strain which contains a ColE1-dnaZ hybrid plasmid.

Centrifugation, Density Gradient↗
PubMed1976

Mutagenic DNA repair in Escherichia coli. III. Requirement for a function of DNA polymerase III in ultraviolet-light mutagenesis.

The polC (= dnaE) temperature-sensitive DNA polymerase III mutation from Escherichia coli BT1026 has been transduced into E. coli WP2 (to give CM731) and WP2 uvr A (to give CM741). In excision-deficient CM741 UV-induced Trp+ mutations progressively lost their photoreversibility during post-irradiation incubation at 34 degrees. Immediately after transfer to 43 degrees, however, there was no further loss of reversibility although post-replication strand joining still occurred and uptake of 3H-thymidine into DNA continued for 20 to 30 min. In excision-proficient CM731, UV lesions capable of leading to Strr mutations disappeared during post-irradiation incubation at restrictive temperature and there was no increase in the number remaining after exposure to photoreversing light. In contrast, at permissive temperature, premutational lesions were not lost and became progressively converted into non-photoreverisble mutations. It is concluded that a function of the polC gene is necessary for error-prone repair to occur and that this function is defective at 43 degrees in the enzyme specified by the polC allele from BT1026. This function seems not to be essential for most post-replication or excision repair or for normal DNA replication and may be particularly involved in the insertion of incorrect bases during error-prone repair.

Coliphages↗
PubMed1978

Bacillus subtilis dnaF: a mutation of the gene specifying the structure of DNA polymerase III.

The characteristics of Bacillus subtilis dnaF, a mutation specifying a temperature sensitive phenotype, were examined to determine its relationship to polC, the gene specifying the structure of DNA polymerase III (pol III). Exposure of growing cells bearing dnaF to non-permissive temperature inhibited replicative DNA synthesis and specifically depressed the expression of pol III activity in crude extracts. Highly purified pol III derived from cells bearing dnaF was temperature.sensitive in its polymerase activity, indicating that dnaF is a specific, polC mutation which specifies a structurally altered enzyme.

Bacillus subtilis↗
PubMed1976

A simplified procedure for the analysis of DNA polymerase III levels in Bacillus subtilis strains.

A simple and reproducible procedure is described which allows the fast and almost quantitative removal of DNA polymerases I and II from DNA polymerase III, in crude extracts of polA+ strains of Bacillus subtilis. The procedure entails streptomycin sulfate and ammonium sulfate fractionations; subsequent analysis of the partially purified preparation by G-200 chromatography, DEAE cellulose chromatography and density gradient sedimentation, shows that the ammonium sulfate fraction contains less than 5% of the total activity as DNA polymerase I and less than 2% as DNA polymerase II. The purification procedure, up to the ammonium sulfate step, was utilized for the analysis of the level of DNA polymerase III in several B. subtilis mutants, with results comparable to those obtained from the corresponding polA- strains following more cumbersome purification procedures. The M.W. of the purified form is of 227.000, somewhat greater than the published values. The early fractions of the purification have revealed the existence of a form with a M.W. of 426.000; the nature of this form, which has been observed in several instances and which is very unstable and short-lived, is under investigation.

Bacillus subtilis↗
PubMed1979

Decreased transfer of pyrimidine dimers from parental to daughter DNA strands in UV-irradiated Escherichia coli deficient in DNA polymerase III.

The number of pyrimidine dimers (sites sensitive to UV-endonuclease from M. luteus) transferred at 43 degrees to daughter DNA strands during postreplication repair in UV-irradiated E. coli uvr A polCts was found to be decreased as compared to that after repair at 32 degrees. This indicates the involvement of DNA polymerase III in the sister DNA recombination in UV-irradiated E. coli.

DNA Polymerase III↗
PubMed1979

Fidelity of replication of phage phi X174 DNA by DNA polymerase III holoenzyme: spontaneous mutation by misincorporation.

DNA from phi X174 is replicated in vitro with a fidelity similar to that found genetically. A mutation of TAG leads to TGG may be induced, however, by varying the concentrations of deoxynucleoside triphosphates, with a frequency proportional to [dGTP]2/[dATP]. This complex concentration dependence is consistent with the active participation of a proofreading mechanism that hydrolytically excises mismatched base pairs as they are formed. A simple kinetic analysis predicts that the frequency of misincorporation depends on the ratio of incorrect to correct deoxynucleoside triphosphates times the concentration of the next triphosphate in the sequence to be added. This suggests that spontaneous mutation by misincorporation depends crucially on the composition of the deoxynucleoside triphosphate pool.

Bacteriophage phi X 174↗
PubMed1977

Inhibitors of Bacillus subtilis DNA polymerase III. Structure-activity relationships of 6-(phenylhydrazino)uracils.

6-(Phenylhydrazino)uracils inhibit the replication-specific enzyme DNA polymerase III of Bacillus subtilis by forming a strong, reversible complex with template-primer DNA and enzyme. The phenyl ring interacts with a hydrophobic enzyme site which, on the basis of structure-activity relationships of substituted analogues, appears to possess the following characteristics: (1) planarity or near-planarity; (2) a finite capacity to accommodate bulky substituents; and (3) location near the domain of the enzyme active site. A mutant DNA polymerase III, derived from a mutant strain of B. subtilis selected for resistance to 6-(p-hydroxyphenylazo)pyrimidines, is resistant only to inhibitors bearing p-hydroxy or amino groups and is hypersensitive to inhibitors containing nonpolar substituents; these results suggest the existence of mutable, secondary regions of the binding site which interact with para substituents and, thus, influence the strength of the primary phenyl-enzyme interaction.

Bacillus subtilis↗
PubMed1976

Mapping of the gene specifying DNA polymerase III of Bacillus subtilis.

polC, the gene specifying the structure of the replication-specific DNA polymerase III of B. subtilis, was mapped by exploiting azp-12, a mutation conferring resistance to azopyrimidine which determines a mutant, azopyrimidine-resistant enzyme. azp-12 was located in the area of the pyrA locus and is between spcB1 and recA1. azp-12 was linked by transformation to four other mutations which influence the in vitro behaviour of DNA polymerase III--polC25, polC26, mut-1(ts), and DNAF133; the close linkage of these five mutations strongly suggests that they are alleles of the same gene.

Azo Compounds↗
PubMed1977

Inhibitors of Bacillus subtilis DNA polymerase III. 6-(arylalkylamino)uracils and 6-anilinouracils.

6-(Benzylamino)uracils and substituted 6-anilinouracils have been found to be potent inhibitors of Bacillus subtilis DNA polymerase III by a mechanism identical with that of 6-(phenylhydrazino)uracils. Higher phenylalkylamino homologues are progressively weaker inhibitors of the enzyme. Examination of the effects of substituents on the activity of 6-(benzylamino)uracils against wild-type and mutant enzymes and preliminary results for 6-anilinouracils have permitted further dissection of the mechanism of inhibition. The experimental results indicate that (1) the polymerase inhibitor binding site is compact, accommodating only small alterations in the distance between the uracil and phenyl rings, (2) the phenyl ring, which provides the major contribution to inhibitor-enzyme binding, adopts a specific active conformation, and (3) an enzyme site which interacts with substituents in the phenyl ring forms a part of the active site of DNA polymerase III.

Aniline Compounds↗