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Results for “DNA Polymerase III”

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At least 19 recordsLinked to original sources
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↗
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↗
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↗
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↗
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↗
PubMed1977

Selective inhibition of in vitro DNA synthesis dependent on phiX174 compared with fd DNA. I. Protein requirements for selective inhibition.

Crude extracts of Escherichia coli selectively convert fd viral DNA and not phiX174 DNA to duplex DNA via a complex series of reactions one of which involves RNA polymerase. Reactions leading to formation of fd duplex-replicative (RFII) structures have been reconstituted with purified proteins from E. coli. Maximal synthesis requires the combined action of E. coli binding protein, DNA elongation factor I, DNA elongation factor II preparations (which are a mixture of dna Z and DNA elongation factor III), DNA polymerase III, DNA-dependent RNA polymerase, Mg2+, dATP, dGTP, dCTP, dTTP, and ATP, GTP, CTP, and UTP. In contrast to crude extracts of E. coli, purified protein fractions do not distinguish between fd DNA and phiX174 DNA in duplex DNA formation. The addition of crude fractions of E. coli to the purified components listed above selectively permits fd RFII formation and prevents phiX RFII formation. This selective inhibition was used as an assay to isolate proteins essential for this phenomenon; they include RNase H, discriminatory factor alpha, and discriminatory factor beta.

Adenosine Triphosphatases↗
PubMed1975

Association of phiX174 DNA-dependent ATPase activity with an Escherichia coli protein, replication factor Y, required for in vitro synthesis of phiX174 DNA.

phiX174 DNA-dependent DNA synthesis is catalyzed in vitro by the combination of at least 11 purified protein fractions: dnaB, dnaC(D), and dnaG gene products, DNA polymerase III, DNA elongation factors I and II, DNA binding protein, and replication factors W, X, Y, and Z. The reaction requires ATP, 4 dNTPs, and Mg+2 and is specific for phiX174 (or phiXahb) DNA. Purified replication factor Y contains phiX174 (or phiXahb) DNA-dependent ATPase (or dATPase) activity. The ATPase activity is poorly stimulated by other single-stranded DNA, by double-stranded DNA, or by RNA. The products of the phiX174 DNA-dependent ATPase activity of factor Y are Pi and ADP (or dADP). The association of phiX174 DNA-dependent ATPase activity with factor Y was shown in the following ways: (a) the two activities copurified with a constant ratio; (b) they comigrated on native polyacrylamide gel electrophoresis; (c) both activities were heat-inactivated at the same rate; and (d) both showed identical patterns of N-ethylmaleimide sensitivity.

Adenosine Triphosphatases↗
PubMed1979

Pyrimidine dimer excision in a Bacillus subtilis Uvr- mutant.

A technique which allows the measurement of small numbers of pyrimidine dimers in the deoxyribonucleic acid (DNA) of cells of Bacillus subtilis irradiated with ultraviolet light has been used to show that a strain mutant at the uvr-1 locus is able to excise pyrimidine dimers. Excision repair in this strain was slow, but incision may not be rate limiting because single-strand breaks in DNA accumulate under some conditions. Excision repair probably accounted for a liquid-holding recovery previously reported to occur in this strain. Recombinational exchange of pyrimidine dimers into newly replicated DNA was readily detected in uvr-1 cells, but this exchange did not account for more than a minor fraction of the dimers removed from parental DNA. Excision repair in the uvr-1 strain was inhibited by a drug which complexes DNA polymerase III with DNA gaps. This inhibition may be limited to a number of sites equal to the number of DNA polymerase III molecules, and it is inferred that large gaps are produced by excision of dimers. Because the uvr-1 mutation specifically interferes with excision of dimers at incision sites, it is concluded that the uvr-1 gene product may be an exonuclease which is essential for efficient dimer excision.

Bacillus subtilis↗
PubMed1978

Isolation of an Escherichia coli K-12 dnaE mutation as a mutator.

Using a papillation method, a large number of Escherichia coli K-12 mutator mutations have been isolated. Only one of these (out of 1,250) mutator mutations has proved to be conditionally lethal at high temperatures. In vivo complementation tests indicated that this mutation, dnaE9, lies in dnaE, the structural gene for DNA polymerase III. The dnaE9 polymerase was not thermolabile in vitro; however, it showed a slow decline in specific activity in vivo at the nonpermissive temperature. Cultures of this mutant exhibited a comparably slow shutoff of DNA synthesis on shift to a nonpermissive temperature. dnaE9 showed temperature-sensitive mutator activity, which is not dependent on recA.

Chromosome Mapping↗