scholarly journals Reduced structural flexibility for an exonuclease deficient DNA polymerase III mutant

2018 ◽  
Vol 20 (42) ◽  
pp. 26892-26902 ◽  
Author(s):  
Hailey L. Gahlon ◽  
Alice R. Walker ◽  
G. Andrés Cisneros ◽  
Meindert H. Lamers ◽  
David S. Rueda
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Structural Flexibility ◽  
Dna Polymerase Iii ◽  
Polymerase Iii ◽  
Speed And Accuracy

DNA synthesis, carried out by DNA polymerases, requires balancing speed and accuracy for faithful replication of the genome.

scholarly journals Antimutator mutations in the alpha subunit of Escherichia coli DNA polymerase III: identification of the responsible mutations and alignment with other DNA polymerases.

Genetics ◽  
1993 ◽  
Vol 134 (4) ◽  
pp. 1039-1044 ◽  
Author(s):  
I J Fijalkowska ◽  
R M Schaaper
Keyword(s):  
Escherichia Coli ◽  
Amino Acid ◽  
Dna Polymerase ◽  
Dna Polymerases ◽  
Alpha Subunit ◽  
Sequence Motifs ◽  
Dna Polymerase Iii ◽  
Conserved Sequence ◽  
Polymerase Iii ◽  
Dna Replication Errors

Abstract The dnaE gene of Escherichia coli encodes the DNA polymerase (alpha subunit) of the main replicative enzyme, DNA polymerase III holoenzyme. We have previously identified this gene as the site of a series of seven antimutator mutations that specifically decrease the level of DNA replication errors. Here we report the nucleotide sequence changes in each of the different antimutator dnaE alleles. For each a single, but different, amino acid substitution was found among the 1,160 amino acids of the protein. The observed substitutions are generally nonconservative. All affected residues are located in the central one-third of the protein. Some insight into the function of the regions of polymerase III containing the affected residues was obtained by amino acid alignment with other DNA polymerases. We followed the principles developed in 1990 by M. Delarue et al. who have identified in DNA polymerases from a large number of prokaryotic and eukaryotic sources three highly conserved sequence motifs, which are suggested to contain components of the polymerase active site. We succeeded in finding these three conserved motifs in polymerase III as well. However, none of the amino acid substitutions responsible for the antimutator phenotype occurred at these sites. This and other observations suggest that the effect of these mutations may be exerted indirectly through effects on polymerase conformation and/or DNA/polymerase interactions.

The Mechanism of Replication of Single-Stranded DNA. VI. Requirements for Ribonucleoside Triphosphates and DNA Polymerase III

1973 ◽  
Vol 51 (12) ◽  
pp. 1588-1597 ◽  
Author(s):  
David T. Denhardt ◽  
Makoto Iwaya ◽  
Grant McFadden ◽  
Gerald Schochetman
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii

Evidence is presented that in Escherichia coli made permeable to nucleotides by exposure to toluene, the synthesis of a DNA chain complementary to the infecting single-stranded DNA of bacteriophage [Formula: see text] requires ATP as well as the four deoxyribonucleoside triphosphates. This synthesis results in the formation of the parental double-stranded replicative-form (RF) molecule. The ATP is not required simply to prevent degradation of the ribonucleoside or deoxyribonucleoside triphosphates; it can be partially substituted for by other ribonucleoside triphosphates.No single one of the known E. coli DNA polymerases appears to be uniquely responsible in vivo for the formation of the parental RF. Since [Formula: see text] replicates well in strains lacking all, or almost all, of the in-vitro activities of DNA polymerases I and II, neither of these two enzymes would seem essential; and in a temperature-sensitive E. coli mutant (dnaEts) deficient in DNA polmerase-I activity and possessing a temperature-sensitive DNA polymerase III, the viral single-stranded DNA is efficiently incorporated into an RF molecule at the restrictive temperature. In contrast, both RF replication and progeny single-stranded DNA synthesis are dependent upon DNA polymerase III activity.

scholarly journals Long-Range PCR Amplification of DNA by DNA Polymerase III Holoenzyme from Thermus thermophilus

2015 ◽  
Vol 2015 ◽  
pp. 1-16 ◽  
Author(s):  
Wendy Ribble ◽  
Shawn D. Kane ◽  
James M. Bullard
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Thermus Thermophilus ◽  
Buffer System ◽  
Dna Polymerase Iii ◽  
Clamp Loader ◽  
Native Form ◽  
Polymerase Iii ◽  
Essential Components ◽  
Pol Iii

DNA replication in bacteria is accomplished by a multicomponent replicase, the DNA polymerase III holoenzyme (pol III HE). The three essential components of the pol III HE are the α polymerase, the β sliding clamp processivity factor, and the DnaX clamp-loader complex. We report here the assembly of the functional holoenzyme from Thermus thermophilus (Tth), an extreme thermophile. The minimal holoenzyme capable of DNA synthesis consists of α, β and DnaX (τ and γ), δ and δ′ components of the clamp-loader complex. The proteins were each cloned and expressed in a native form. Each component of the system was purified extensively. The minimum holoenzyme from these five purified subunits reassembled is sufficient for rapid and processive DNA synthesis. In an isolated form the α polymerase was found to be unstable at temperatures above 65°C. We were able to increase the thermostability of the pol III HE to 98°C by addition and optimization of various buffers and cosolvents. In the optimized buffer system we show that a replicative polymerase apparatus, Tth pol III HE, is capable of rapid amplification of regions of DNA up to 15,000 base pairs in PCR reactions.

scholarly journals Processive DNA synthesis by DNA polymerase II mediated by DNA polymerase III accessory proteins.

1992 ◽  
Vol 267 (16) ◽  
pp. 11431-11438
Author(s):  
C.A. Bonner ◽  
P.T. Stukenberg ◽  
M Rajagopalan ◽  
R Eritja ◽  
M O'Donnell ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Accessory Proteins ◽  
Dna Polymerase Iii ◽  
Polymerase Iii ◽  
Dna Polymerase Ii

scholarly journals The Structure of T. aquaticus DNA Polymerase III Is Distinct from Eukaryotic Replicative DNA Polymerases

Cell ◽  
2006 ◽  
Vol 126 (5) ◽  
pp. 893-904 ◽  
Author(s):  
Scott Bailey ◽  
Richard A. Wing ◽  
Thomas A. Steitz
Keyword(s):  
Dna Polymerase ◽  
Dna Polymerases ◽  
Dna Polymerase Iii ◽  
Polymerase Iii
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