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.

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.

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.

Genetic Analysis of Yeast RPA1 Reveals Its Multiple Functions in DNA Metabolism

Genetics ◽  
1998 ◽  
Vol 148 (3) ◽  
pp. 989-1005 ◽  
Author(s):  
Keiko Umezu ◽  
Neal Sugawara ◽  
Clark Chen ◽  
James E Haber ◽  
Richard D Kolodner
Keyword(s):  
Dna Replication ◽  
Dna Binding ◽  
Protein A ◽  
Temperature Sensitive ◽  
Restrictive Temperature ◽  
Physical Analysis ◽  
Single Stranded Dna ◽  
Temperature Sensitive Mutants

Abstract Replication protein A (RPA) is a single-stranded DNA-binding protein identified as an essential factor for SV40 DNA replication in vitro. To understand the in vivo functions of RPA, we mutagenized the Saccharomyces cerevisiae RFA1 gene and identified 19 ultraviolet light (UV) irradiation- and methyl methane sulfonate (MMS)-sensitive mutants and 5 temperature-sensitive mutants. The UV- and MMS-sensitive mutants showed up to 104 to 105 times increased sensitivity to these agents. Some of the UV- and MMS-sensitive mutants were killed by an HO-induced double-strand break at MAT. Physical analysis of recombination in one UV- and MMS-sensitive rfa1 mutant demonstrated that it was defective for mating type switching and single-strand annealing recombination. Two temperature-sensitive mutants were characterized in detail, and at the restrictive temperature were found to have an arrest phenotype and DNA content indicative of incomplete DNA replication. DNA sequence analysis indicated that most of the mutations altered amino acids that were conserved between yeast, human, and Xenopus RPA1. Taken together, we conclude that RPA1 has multiple roles in vivo and functions in DNA replication, repair, and recombination, like the single-stranded DNA-binding proteins of bacteria and phages.

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 INTERACTION OF DNA POLYMERASE III γ AND β SUBUNITS IN VIVO IN SALMONELLA TYPHIMURIUM

Genetics ◽  
1986 ◽  
Vol 113 (3) ◽  
pp. 499-515
Author(s):  
Joyce Engstrom ◽  
Annette Wong ◽  
Russell Maurer
Keyword(s):  
Salmonella Typhimurium ◽  
Dna Polymerase ◽  
Regulatory Gene ◽  
Temperature Sensitive ◽  
Physical Interaction ◽  
Dna Polymerase Iii ◽  
Β Subunit ◽  
Γ Subunit ◽  
Polymerase Iii

ABSTRACT We show that temperature-sensitive mutations in dnaZ, the gene for the γ subunit of DNA polymerase III holoenzyme, can be suppressed by mutations in the dnaN gene, which encodes the β subunit. These results support a direct physical interaction of these two subunits during polymerase assembly or function. The suppressor phenotype is also sensitive to modulation by the dnaA genotype. Since dnaA is organized in an operon with dnaN, and dnaA is a regulatory gene of this operon, we propose that the dnaA effect on suppression can best be explained by modulation of suppressor dnaN levels.

scholarly journals Distinct Double- and Single-Stranded DNA Binding of E. coli Replicative DNA Polymerase III α Subunit

2008 ◽  
Vol 3 (9) ◽  
pp. 577-587 ◽  
Author(s):  
Micah J. McCauley ◽  
Leila Shokri ◽  
Jana Sefcikova ◽  
Česlovas Venclovas ◽  
Penny J. Beuning ◽  
...  
Keyword(s):  
Dna Binding ◽  
Dna Polymerase ◽  
Dna Polymerase Iii ◽  
Single Stranded Dna ◽  
Α Subunit ◽  
E Coli ◽  
Polymerase Iii

scholarly journals Suppression of Temperature-Sensitive Chromosome Replication of an Escherichia coli dnaX(Ts) Mutant by Reduction of Initiation Efficiency

2003 ◽  
Vol 185 (12) ◽  
pp. 3583-3595 ◽  
Author(s):  
Alexandra Blinkova ◽  
Mary Jo Hermandson ◽  
James R. Walker
Keyword(s):  
Dna Polymerase ◽  
Temperature Sensitivity ◽  
Temperature Sensitive ◽  
Dna Polymerase Iii ◽  
Suppressor Mutations ◽  
Polymerase Iii ◽  
Initiation Frequency ◽  
Cold Sensitive ◽  
Ts Mutant

ABSTRACT Temperature sensitivity of DNA polymerization and growth of a dnaX(Ts) mutant is suppressible at 39 to 40°C by mutations in the initiator gene, dnaA. These suppressor mutations concomitantly cause initiation inhibition at 20°C and have been designated Cs,Sx to indicate both phenotypic characteristics of cold-sensitive initiation and suppression of dnaX(Ts). One dnaA(Cs,Sx) mutant, A213D, has reduced affinity for ATP, and two mutants, R432L and T435K, have eliminated detectable DnaA box binding in vitro. Two models have explained dnaA(Cs,Sx) suppression of dnaX, which codes for both the τ and γ subunits of DNA polymerase III. The initiation deficiency model assumes that reducing initiation efficiency allows survival of the dnaX(Ts) mutant at the somewhat intermediate temperature of 39 to 40°C by reducing chromosome content per cell, thus allowing partially active DNA polymerase III to complete replication of enough chromosomes for the organism to survive. The stabilization model is based on the idea that DnaA interacts, directly or indirectly, with polymerization factors during replication. We present five lines of evidence consistent with the initiation deficiency model. First, a dnaA(Cs,Sx) mutation reduced initiation frequency and chromosome content (measured by flow cytometry) and origin/terminus ratios (measured by real-time PCR) in both wild-type and dnaX(Ts) strains growing at 39 and 34°C. These effects were shown to result specifically from the Cs,Sx mutations, because the dnaX(Ts) mutant is not defective in initiation. Second, reduction of the number of origins and chromosome content per cell was common to all three known suppressor mutations. Third, growing the dnaA(Cs,Sx) dnaX(Ts) strain on glycerol-containing medium reduced its chromosome content to one per cell and eliminated suppression at 39°C, as would be expected if the combination of poor carbon source, the Cs,Sx mutation, the Ts mutation, and the 39°C incubation reduced replication to the point that growth (and, therefore, suppression) was not possible. However, suppression was possible on glycerol medium at 38°C. Fourth, the dnaX(Ts) mutation can be suppressed also by introduction of oriC mutations, which reduced initiation efficiency and chromosome number per cell, and the degree of suppression was proportional to the level of initiation defect. Fifth, introducing a dnaA(Cos) allele, which causes overinitiation, into the dnaX(Ts) mutant exacerbated its temperature sensitivity.

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.

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