scholarly journals Dysfunctional proofreading in the Escherichia coli DNA polymerase III core

2004 ◽  
Vol 384 (2) ◽  
pp. 337-348 ◽  
Author(s):  
Duane A. LEHTINEN ◽  
Fred W. PERRINO
Keyword(s):  
Dna Polymerase ◽  
Gel Filtration ◽  
Exonuclease Activity ◽  
Dna Polymerase Iii ◽  
Wild Type ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

The ε-subunit contains the catalytic site for the 3′→5′ proofreading exonuclease that functions in the DNA pol III (DNA polymerase III) core to edit nucleotides misinserted by the α-subunit DNA pol. A novel mutagenesis strategy was used to identify 23 dnaQ alleles that exhibit a mutator phenotype in vivo. Fourteen of the ε mutants were purified, and these proteins exhibited 3′→5′ exonuclease activities that ranged from 32% to 155% of the activity exhibited by the wild-type ε protein, in contrast with the 2% activity exhibited by purified MutD5 protein. DNA pol III core enzymes constituted with 11 of the 14 ε mutants exhibited an increased error rate during in vitro DNA synthesis using a forward mutation assay. Interactions of the purified ε mutants with the α- and θ-subunits were examined by gel filtration chromatography and exonuclease stimulation assays, and by measuring polymerase/exonuclease ratios to identify the catalytically active ε511 (I170T/V215A) mutant with dysfunctional proofreading in the DNA pol III core. The ε511 mutant associated tightly with the α-subunit, but the exonuclease activity of ε511 was not stimulated in the α–ε511 complex. Addition of the θ-subunit to generate the α–ε511–θ DNA pol III core partially restored stimulation of the ε511 exonuclease, indicating a role for the θ-subunit in co-ordinating the α–ε polymerase–exonuclease interaction. The α–ε511–θ DNA pol III core exhibited a 3.5-fold higher polymerase/exonuclease ratio relative to the wild-type DNA pol III core, further indicating dysfunctional proofreading in the α–ε511–θ complex. Thus the ε511 mutant has wild-type 3′→5′ exonuclease activity and associates physically with the α- and θ-subunits to generate a proofreading-defective DNA pol III enzyme.

scholarly journals In Vivo Protein Interactions within theEscherichia coli DNA Polymerase III Core

1998 ◽  
Vol 180 (6) ◽  
pp. 1563-1566 ◽  
Author(s):  
Piotr Jonczyk ◽  
Adrianna Nowicka ◽  
Iwona J. Fijałkowska ◽  
Roel M. Schaaper ◽  
Zygmunt Cieśla
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Physiological Role ◽  
Dna Polymerase Iii ◽  
Mutator Phenotype ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Pol Iii

ABSTRACT The mechanisms that control the fidelity of DNA replication are being investigated by a number of approaches, including detailed kinetic and structural studies. Important tools in these studies are mutant versions of DNA polymerases that affect the fidelity of DNA replication. It has been suggested that proper interactions within the core of DNA polymerase III (Pol III) of Escherichia colicould be essential for maintaining the optimal fidelity of DNA replication (H. Maki and A. Kornberg, Proc. Natl. Acad. Sci. USA 84:4389–4392, 1987). We have been particularly interested in elucidating the physiological role of the interactions between the DnaE (α subunit [possessing DNA polymerase activity]) and DnaQ (ɛ subunit [possessing 3′→5′ exonucleolytic proofreading activity]) proteins. In an attempt to achieve this goal, we have used theSaccharomyces cerevisiae two-hybrid system to analyze specific in vivo protein interactions. In this report, we demonstrate interactions between the DnaE and DnaQ proteins and between the DnaQ and HolE (θ subunit) proteins. We also tested the interactions of the wild-type DnaE and HolE proteins with three well-known mutant forms of DnaQ (MutD5, DnaQ926, and DnaQ49), each of which leads to a strong mutator phenotype. Our results show that the mutD5 anddnaQ926 mutations do not affect the ɛ subunit-α subunit and ɛ subunit-θ subunit interactions. However, thednaQ49 mutation greatly reduces the strength of interaction of the ɛ subunit with both the α and the θ subunits. Thus, the mutator phenotype of dnaQ49 may be the result of an altered conformation of the ɛ protein, which leads to altered interactions within the Pol III core.

The mutant β E202K sliding clamp protein impairs DNA polymerase III replication activity

2021 ◽  
Author(s):  
Caleb Homiski ◽  
Michelle K. Scotland ◽  
Vignesh M. P. Babu ◽  
Sundari Chodavarapu ◽  
Robert W. Maul ◽  
...  
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Genetic Selection ◽  
Cellular Level ◽  
Dna Polymerase Iii ◽  
E Coli ◽  
Polymerase Iii ◽  
Pol Iii

Expression of the E. coli dnaN -encoded β clamp at ≥10-fold higher than chromosomally-expressed levels impedes growth by interfering with DNA replication. We hypothesized that the excess β clamp sequesters the replicative DNA polymerase III (Pol III) to inhibit replication. As a test of this hypothesis, we measured the ability of eight mutant clamps obtained by their inability to impede growth to stimulate Pol III replication in vitro . Compared with the wild type clamp, seven of the mutants were defective, consistent with their elevated cellular levels failing to sequester Pol III. However, the β E202K mutant, which bears a glutamic acid-to-lysine substitution at residue 202 displayed an increased affinity for Pol IIIα and Pol III core (Pol IIIαεθ), suggesting that it could still effectively sequester Pol III. Of interest, β E202K supported in vitro DNA replication by Pol II and Pol IV, but was defective with Pol III. Genetic experiments indicated that the dnaN E202K strain remained proficient in DNA damage-induced mutagenesis, but was modestly induced for SOS and displayed sensitivity to ultraviolet light and methyl methanesulfonate. These results correlate an impaired ability of the mutant β E202K clamp to support Pol III replication in vivo with its in vitro defect in DNA replication. Taken together, our results: (i) support the model that sequestration of Pol III contributes to growth inhibition, (ii) argue for existence of an additional mechanism that contributes to lethality and (iii) suggest that physical and functional interactions of the β clamp with Pol III are more extensive than currently appreciated. IMPORTANCE The β clamp plays critically important roles in managing the actions of multiple proteins at the replication fork. However, we lack a molecular understanding of both how the clamp interacts with these different partners, and the mechanisms by which it manages their respective actions. We previously exploited the finding that an elevated cellular level of the β clamp impedes E. coli growth by interfering with DNA replication. Using a genetic selection method, we obtained novel mutant β clamps that fail to inhibit growth. Their analysis revealed that β E202K is unique among them. Our work offers new insights into how the β clamp interacts with and manages the actions of E. coli DNA polymerases II, III and IV.

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 The θ Subunit of Escherichia coli DNA Polymerase III: a Role in Stabilizing the ε Proofreading Subunit

2004 ◽  
Vol 186 (9) ◽  
pp. 2774-2780 ◽  
Author(s):  
Sharon A. Taft-Benz ◽  
Roel M. Schaaper
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Normal Cell ◽  
Mutation Rates ◽  
Dna Polymerase Iii ◽  
Α Subunit ◽  
Polymerase Iii ◽  
Separate Protein ◽  
Gram Negative Organisms

ABSTRACT The function of the θ subunit of Escherichia coli DNA polymerase III holoenzyme is not well established. θ is a tightly bound component of the DNA polymerase III core, which contains the α subunit (polymerase), the ε subunit (3′→5′ exonuclease), and the θ subunit, in the linear order α-ε-θ. Previous studies have shown that the θ subunit is not essential, as strains carrying a deletion of the holE gene (which encodes θ) proved fully viable. No significant phenotypic effects of the holE deletion could be detected, as the strain displayed normal cell health, morphology, and mutation rates. On the other hand, in vitro experiments have indicated the efficiency of the 3′-exonuclease activity of ε to be modestly enhanced by the presence of θ. Here, we report a series of genetic experiments that suggest that θ has a stabilizing role for the ε proofreading subunit. The observations include (i) defined ΔholE mutator effects in mismatch-repair-defective mutL backgrounds, (ii) strong ΔholE mutator effects in certain proofreading-impaired dnaQ strains, and (iii) yeast two- and three-hybrid experiments demonstrating enhancement of α-ε interactions by the presence of θ. θ appears conserved among gram-negative organisms which have an exonuclease subunit that exists as a separate protein (i.e., not part of the polymerase polypeptide), and the presence of θ might be uniquely beneficial in those instances where the proofreading 3′-exonuclease is not part of the polymerase polypeptide.

scholarly journals Escherichia coli DNA Polymerase III τ- and γ-Subunit Conserved Residues Required for Activity In Vivo and In Vitro

2000 ◽  
Vol 182 (21) ◽  
pp. 6106-6113 ◽  
Author(s):  
James R. Walker ◽  
Christine Hervas ◽  
Julie D. Ross ◽  
Alexandra Blinkova ◽  
Michael J. Walbridge ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Atpase Activity ◽  
Dna Polymerase ◽  
Mutant Gene ◽  
Dna Polymerase Iii ◽  
Conserved Residues ◽  
Polymerase Iii ◽  
Clamp Loading

ABSTRACT The Escherichia coli DNA polymerase III τ and γ subunits are single-strand DNA-dependent ATPases (the latter requires the δ and δ′ subunits for significant ATPase activity) involved in loading processivity clamp β. They are homologous to clamp-loading proteins of many organisms from phages to humans. Alignment of 27 prokaryotic τ/γ homologs and 1 eukaryotic τ/γ homolog has refined the sequences of nine previously defined identity and functional motifs. Mutational analysis has defined highly conserved residues required for activity in vivo and in vitro. Specifically, mutations introduced into highly conserved residues within three of those motifs, the P loop, the DExx region, and the SRC region, inactivated complementing activity in vivo and clamp loading in vitro and reduced ATPase catalytic efficiency in vitro. Mutation of a highly conserved residue within a fourth motif, VIc, inactivated clamp-loading activity and reduced ATPase activity in vitro, but the mutant gene, on a multicopy plasmid, retained complementing activity in vivo and the mutant gene also supported apparently normal replication and growth as a haploid, chromosomal allele.

scholarly journals Stabilization of the Escherichia coli DNA polymerase III ε subunit by the θ subunit favors in vivo assembly of the Pol III catalytic core

2012 ◽  
Vol 523 (2) ◽  
pp. 135-143 ◽  
Author(s):  
Emanuele Conte ◽  
Gabriele Vincelli ◽  
Roel M. Schaaper ◽  
Daniela Bressanin ◽  
Alessandra Stefan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Polymerase ◽  
Dna Polymerase Iii ◽  
Catalytic Core ◽  
Polymerase Iii ◽  
Pol Iii

scholarly journals E. coli primase and DNA polymerase III holoenzyme are able to bind concurrently to a primed template during DNA replication

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Andrea Bogutzki ◽  
Natalie Naue ◽  
Lidia Litz ◽  
Andreas Pich ◽  
Ute Curth
Keyword(s):  
Dna Replication ◽  
Dna Polymerase ◽  
Protein Interactions ◽  
Dna Polymerase Iii ◽  
Protein Protein Interactions ◽  
Single Stranded Dna ◽  
E Coli ◽  
Polymerase Iii ◽  
C Terminus ◽  
Pol Iii

Abstract During DNA replication in E. coli, a switch between DnaG primase and DNA polymerase III holoenzyme (pol III) activities has to occur every time when the synthesis of a new Okazaki fragment starts. As both primase and the χ subunit of pol III interact with the highly conserved C-terminus of single-stranded DNA-binding protein (SSB), it had been proposed that the binding of both proteins to SSB is mutually exclusive. Using a replication system containing the origin of replication of the single-stranded DNA phage G4 (G4ori) saturated with SSB, we tested whether DnaG and pol III can bind concurrently to the primed template. We found that the addition of pol III does not lead to a displacement of primase, but to the formation of higher complexes. Even pol III-mediated primer elongation by one or several DNA nucleotides does not result in the dissociation of DnaG. About 10 nucleotides have to be added in order to displace one of the two primase molecules bound to SSB-saturated G4ori. The concurrent binding of primase and pol III is highly plausible, since even the SSB tetramer situated directly next to the 3′-terminus of the primer provides four C-termini for protein-protein interactions.

scholarly journals In vitro analysis of elongation and termination by mutant RNA polymerases with altered termination behavior.

1996 ◽  
Vol 16 (11) ◽  
pp. 6468-6476 ◽  
Author(s):  
S A Shaaban ◽  
E V Bobkova ◽  
D M Chudzik ◽  
B D Hall
Keyword(s):  
Rna Polymerase Iii ◽  
Multiple Roles ◽  
Wild Type ◽  
Protein Motifs ◽  
Study Support ◽  
Vitro Analysis ◽  
Pol Iii ◽  
In Vitro Analysis

We have studied the in vitro elongation and termination properties of several yeast RNA polymerase III (pol III) mutant enzymes that have altered in vivo termination behavior (S. A. Shaaban, B. M. Krupp, and B. D. Hall, Mol. Cell. Biol. 15:1467-1478, 1995). The pattern of completed-transcript release was also characterized for three of the mutant enzymes. The mutations studied occupy amino acid regions 300 to 325, 455 to 521, and 1061 to 1082 of the RET1 protein (P. James, S. Whelen, and B. D. Hall, J. Biol. Chem. 266:5616-5624, 1991), the second largest subunit of yeast RNA pol III. In general, mutant enzymes which have increased termination require a longer time to traverse a template gene than does wild-type pol III; the converse holds true for most decreased-termination mutants. One increased-termination mutant (K310T I324K) was faster and two reduced termination mutants (K512N and T455I E478K) were slower than the wild-type enzyme. In most cases, these changes in overall elongation kinetics can be accounted for by a correspondingly longer or shorter dwell time at pause sites within the SUP4 tRNA(Tyr) gene. Of the three mutants analyzed for RNA release, one (T455I) was similar to the wild type while the two others (T455I E478K and E478K) bound the completed SUP4 pre-tRNA more avidly. The results of this study support the view that termination is a multistep pathway in which several different regions of the RET1 protein are actively involved. Region 300 to 325 likely affects a step involved in RNA release, while the Rif homology region, amino acids 455 to 521, interacts with the nascent RNA 3' end. The dual effects of several mutations on both elongation kinetics and RNA release suggest that the protein motifs affected by them have multiple roles in the steps leading to transcription termination.

Sign in / Sign up

Export Citation Format

Share Document