Identification of the Acidic Residues in the Active Site of DNA Polymerase III

Pritchard, A. E.; McHenry, Charles S.

doi:10.1006/jmbi.1998.2352

Cited by 46 publications

(62 citation statements)

References 53 publications

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“…Characteristics of dnaE mutants. Until the recent discovery of auxiliary polymerases, the DNA polymerases were classified into four families (30): polymerases homologous to E. coli pol I, eukaryotic pol alpha, eukaryotic pol beta and terminal transferase, and E. coli pol III. The pol III family is the sole member without a crystallized representative and until recently had few if any recognized homologs.…”

Section: Resultsmentioning

confidence: 99%

“…The pol III family is the sole member without a crystallized representative and until recently had few if any recognized homologs. The rapid progress in whole-genome sequencing has resulted in the identification of numerous microbial homologs (30). We used the Multalin program (4) to align 13 polymerases in order to ascertain the possible importance of the mutator sites and to compare their locations with the active-site aspartic acid residues identified at E. coli dnaE codons 401, 403, and 555 (30).…”

Section: Resultsmentioning

confidence: 99%

“…The rapid progress in whole-genome sequencing has resulted in the identification of numerous microbial homologs (30). We used the Multalin program (4) to align 13 polymerases in order to ascertain the possible importance of the mutator sites and to compare their locations with the active-site aspartic acid residues identified at E. coli dnaE codons 401, 403, and 555 (30). The three mutators that we identified and the previously reported dnaE173 (E612K) (22) were all located by the program at highly conserved sites (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Role of thedinBGene Product in Spontaneous Mutation inEscherichia coliwith an Impaired Replicative Polymerase

et al. 2000

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We isolated several new mutator mutations of the Escherichia coli replicative polymerase dnaE subunit alpha and used them and a previously reported dnaE mutation to study spontaneous frameshift and base substitution mutations. Two of these dnaE strains produce many more mutants when grown on rich (Luria-Bertani) than on minimal medium. A differential effect of the medium was not observed when these dnaE mutations were combined with a mismatch repair mutation. The selection scheme for the dnaE mutations required that they be able to complement a temperature-sensitive strain. However, the ability to complement is not related to the mutator effect for at least one of the mutants. Comparison of the mutation rates for frameshift and base substitution mutations in mutS and dnaE mutS strains suggests that the mismatch repair proteins respond differently to the two types of change. Deletion of dinB from both chromosome and plasmid resulted in a fourto fivefold decrease in the rate of frameshift and base substitution mutations in a dnaE mutS double mutant background. This reduction indicates that most mistakes in replication occur as a result of the action of the auxiliary rather than the replicative polymerase in this dnaE mutant. Deletion of dinB from strains carrying a wild-type dnaE had a measurable effect, suggesting that a fraction of spontaneous mutations occur as a result of dinB polymerase action even in cells with a normal replicative polymerase.Mutation is a characteristic of all living systems and provides the material for natural selection (43, 48). However, most mutations are deleterious, and organisms have evolved mechanisms to protect themselves from excessive mutation rates. These protective mechanisms recognize and correct mismatches that have occurred in DNA as a result of replication or spontaneous deamination and recognize and remove potentially mutagenic changes that have occurred as a result of the reaction of the DNA with endogenous or exogenous mutagens (21). The activity of these repair systems can be modulated, and one method of increasing the rate of mutations either permanently or transiently is to decrease the activity of the repair systems, e.g., mismatch repair (14). It has generally been assumed that the interaction of the error repair systems with the natural error rate of the replicative DNA polymerases satisfactorily accounts for mutation rates and their modulation (7). However, among the characteristics of biological systems are their complexity and the multilevels of control that they employ. Regulation, for example, is characterized by both accelerating and inhibiting or braking features, and this principle can be seen in systems as diverse as the regulation of the rate of the heartbeat in vertebrates (38) and the regulation of lactose utilization in bacteria (31). The recent discoveries of DNA polymerases which seem designed to produce a high frequency of errors should therefore not be viewed with surprise, but rather as another illustration of the redundancy with which organisms m...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Role of thedinBGene Product in Spontaneous Mutation inEscherichia coliwith an Impaired Replicative Polymerase

et al. 2000

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“…Its central polymerase domain contains three essential catalytic residues that are presumably required to bind the two Mg 2ϩ ions required for catalysis in other polymerases (28) (Fig. 2).…”

Section: Resultsmentioning

confidence: 99%

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

Wieczorek

McHenry

2006

Journal of Biological Chemistry

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The ␣ subunit of the replicase of all bacteria contains a php domain, initially identified by its similarity to histidinol phosphatase but of otherwise unknown function (Aravind, L., and Koonin, E. V. (1998) Nucleic Acids Res. 26, 3746 -3752). Deletion of 60 residues from the NH 2 terminus of the ␣ php domain destroys ⑀ binding. The minimal 255-residue php domain, estimated by sequence alignment with homolog YcdX, is insufficient for ⑀ binding. However, a 320-residue segment including sequences that immediately precede the polymerase domain binds ⑀ with the same affinity as the 1160-residue full-length ␣ subunit. A subset of mutations of a conserved acidic residue (Asp 43 in Escherichia coli ␣) present in the php domain of all bacterial replicases resulted in defects in ⑀ binding. Using sequence alignments, we show that the prototypical Gram(؉) Pol C, which contains the polymerase and proofreading activities within the same polypeptide chain, has an ⑀-like sequence inserted in a surface loop near the center of the homologous YcdX protein. These findings suggest that the php domain serves as a platform to enable coordination of proofreading and polymerase activities during chromosomal replication.The initial discovery of a proofreading 3Ј35Ј exonuclease within DNA polymerase I provided the prototype for proofreading among all polymerases (1). Editing activity was found in the same polypeptide chain as Pol 2 I and required exchange of the 3Ј-primer terminus between the polymerase and proofreading exonuclease sites, which are separated by ϳ30 Å (2). The structure of the exonuclease site and functional studies with cross-linked substrates suggested that ϳ4 bases needed to be unwound to permit migration of the 3Ј end of a primer from the Pol to exonuclease active site (2, 3). This provided a basis for the preference of the 3Ј35Ј exonuclease for mispaired over properly base-paired primer termini. Partitioning of the primer terminus between the exonuclease and polymerase sites is determined by both DNA structure and protein contacts (4). The kinetic basis for efficient proofreading is the very slow rate of elongation of primers containing a terminal mismatch, providing time for partitioning (5, 6). The exonucleolytic mechanism requires two Mg 2ϩ ions tethered by acidic side chains within the polymerase active site (2), which demonstrates the close functional relationship between the two activities. Escherichia coli Pol II also contains an exonuclease activity in the same polypeptide chain as the polymerase (7).The multisubunit core of the E. coli Pol III replicase was found to contain the polymerase subunit ␣ bound to subunits ⑀ and (8). ⑀ is the product of the mutD gene and was initially identified as conferring high levels of spontaneous mutagenesis when defective (9). In contrast to Pol I and II, ⑀ was shown to be the separate proofreading subunit of the Pol III replicase (10 -13). Holoenzyme reconstituted with ⑀ also exhibits lower fidelity in vitro (11,14).Similar proofreading activities are found in eukaryotic nu...

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“…One approach has been to identify conserved aspartate residues, since carboxylate residues are an essential feature of the polymerase active centers of DNA and RNA polymerases (3,4,14,15). Protein sequence alignment of the ␣-subunit sequences from Proteobacteria, Spirochaetales, Cyanobacteria, Aquificales, and Fermicutes revealed conserved aspartate residues, and three of these residues, D401, D403, and D555, in the E. coli ␣ subunit were determined to be essential for polymerase activity by site-directed mutagenesis (22).…”

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Identification of Escherichia coli dnaE ( polC ) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine

Hiratsuka

Reha-Krantz

2000

J Bacteriol

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Bacteria with reduced DNA polymerase I activity have increased sensitivity to killing by chain-terminating nucleotides (S. A. Rashbaum and N. R. Cozzarelli, Nature 264:679-680, 1976). We have used this observation as the basis of a genetic strategy to identify mutations in the dnaE (polC) gene of Escherichia coli that alter sensitivity to 2,3-dideoxyadenosine (ddA). Two dnaE (polC) mutant strains with increased sensitivity to ddA and one strain with increased resistance were isolated and characterized. The mutant phenotypes are due to single amino acid substitutions in the ␣ subunit, the protein product of the dnaE (polC) gene. Increased sensitivity to ddA is produced by the L329F and H417Y substitutions, and increased resistance is produced by the G365S substitution. The L329F and H417Y substitutions also reduce the accuracy of DNA replication (the mutator phenotype), while the G365S substitution increases accuracy (the antimutator phenotype). All of the amino acid substitutions are in conserved regions near essential aspartate residues. These results prove the effectiveness of the genetic strategy in identifying informative dnaE (polC) mutations that can be used to elucidate the molecular basis of nucleotide interactions in the ␣ subunit of the DNA polymerase III holoenzyme.Escherichia coli DNA polymerase III (DNA Pol III) is the enzyme responsible for replication of the bacterial chromosome. The DNA Pol III holoenzyme consists of 10 subunits, with polymerase activity residing in the ␣ subunit, the product of the dnaE (polC) gene (reviewed in references 16 and 20). Although much is known about the polymerase active centers of several DNA polymerases and an RNA polymerase from structural and genetic studies (14), relatively little is known about the polymerase active centers of bacterial DNA Pol III holoenzymes. One approach has been to identify conserved aspartate residues, since carboxylate residues are an essential feature of the polymerase active centers of DNA and RNA polymerases (3,4,14,15). Protein sequence alignment of the ␣-subunit sequences from Proteobacteria, Spirochaetales, Cyanobacteria, Aquificales, and Fermicutes revealed conserved aspartate residues, and three of these residues, D401, D403, and D555, in the E. coli ␣ subunit were determined to be essential for polymerase activity by site-directed mutagenesis (22).Genetic selection and screening procedures have also been used to identify amino acid residues in the ␣ subunits of bacterial DNA Pol III holoenzymes that are important for function. One advantage of genetic strategies is that structural information is not required, and a second advantage is that the mutant DNA polymerases are studied in the cell in the presence of the full complement of DNA replication proteins. Fijalkowska et al. (7) used a genetic screen to identify mutations in the E. coli dnaE (polC) gene that confer an antimutator phenotype, which is increased accuracy of DNA replication. A mutation that decreases replication fidelity and produces a strong mutator phenotype has a...

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Identification of the Acidic Residues in the Active Site of DNA Polymerase III

Cited by 46 publications

References 53 publications

Role of thedinBGene Product in Spontaneous Mutation inEscherichia coliwith an Impaired Replicative Polymerase

Role of thedinBGene Product in Spontaneous Mutation inEscherichia coliwith an Impaired Replicative Polymerase

The NH2-terminal php Domain of the α Subunit of the Escherichia coli Replicase Binds the ϵ Proofreading Subunit

Identification of Escherichia coli dnaE ( polC ) Mutants with Altered Sensitivity to 2′,3′-Dideoxyadenosine

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