The reversion of trp frameshift mutations in mut, polA, lig and dnaE mutant strains of Escherichia coli

Siegel, Eli C.; Vaccaro, Karen K.

doi:10.1016/0027-5107(78)90055-6

Cited by 40 publications

(19 citation statements)

References 22 publications

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“…The two lac alleles permit specific measurement of a G ⅐ C3T ⅐ A or A ⅐ T3T ⅐ A transversion using the FЈprolac originally present in strains CC104 or CC105 (9). The trpE9777 allele permits measurement of (Ϫ1) frameshift errors by loss of an A ⅐ T base pair from a run of six A ⅐ T pairs (75). Rifampin resistance measures a variety of base substitutions in the rpoB gene (26).…”

Section: Resultsmentioning

confidence: 99%

Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli : Analysis of the dnaX36 Mutator Mutant

et al. 2008

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The dnaX36(TS) mutant of Escherichia coli confers a distinct mutator phenotype characterized by enhancement of transversion base substitutions and certain (؊1) frameshift mutations. Here, we have further investigated the possible mechanism(s) underlying this mutator effect, focusing in particular on the role of the various E. coli DNA polymerases. The dnaX gene encodes the subunit of DNA polymerase III (Pol III) holoenzyme, the enzyme responsible for replication of the bacterial chromosome. The dnaX36 defect resides in the C-terminal domain V of , essential for interaction of with the ␣ (polymerase) subunit, suggesting that the mutator phenotype is caused by an impaired or altered ␣-interaction. We previously proposed that the mutator activity results from aberrant processing of terminal mismatches created by Pol III insertion errors. The present results, including lack of interaction of dnaX36 with mutM, mutY, and recA defects, support our assumption that dnaX36-mediated mutations originate as errors of replication rather than DNA damagerelated events. Second, an important role is described for DNA Pol II and Pol IV in preventing and producing, respectively, the mutations. In the system used, a high fraction of the mutations is dependent on the action of Pol IV in a (dinB) gene dosage-dependent manner. However, an even larger but opposing role is deduced for Pol II, revealing Pol II to be a major editor of Pol III mediated replication errors. Overall, the results provide insight into the interplay of the various DNA polymerases, and of subunit, in securing a high fidelity of replication.The mechanisms by which cells produce mutations, or try to avoid making them, are of significant research interest. Mutations may occur from replication errors, as DNA replication proceeds with high but not infinite accuracy. While the fidelity of individual DNA polymerases, including their base insertion fidelity and proofreading ability, has been investigated in detail (for reviews, see references 43 and 44), recent emphasis has shifted to the fidelity of the chromosomal replisomes, the multisubunit complexes that perform the simultaneous replication of leading and lagging strands. Specific issues of interest are the contribution of the various replisomal subunits, the mechanisms underlying the differential fidelity of leading and lagging strand replication, and the involvement of the additional DNA polymerases that have been discovered in recent years.In the model system Escherichia coli, chromosomal replication is performed by the 17-subunit protein complex DNA polymerase III (Pol III) holoenzyme (HE) (49,50,56). HE is organized into several functional modules: two Pol III core units (one for each strand), two ␤-clamp processivity factors, and the DnaX complex. Each Pol III core is made up of three subunits (␣, ε, and ), in which ␣ is the DNA polymerase, ε is the proofreading subunit (3Ј35Ј exonuclease), and is a stabilizing factor for the ε subunit (37, 82). Each ␤-clamp is a dimer of identical subunits (␤ 2 ) in the shape ...

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Section: Resultsmentioning

confidence: 99%

Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli : Analysis of the dnaX36 Mutator Mutant

et al. 2008

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“…The MMR pathway recognizes and repairs DNA polymerase errors, contributing to the overall fidelity of the DNA replication pathway (reviewed in references 42 and 60). In both E. coli and B. subtilis, deletion of the genes mutS and mutL increases the spontaneous mutation frequency several hundredfold (13,25,63). In E. coli, MutS recognizes and binds mismatches, while MutL functions as a "matchmaker," coordinating the actions of other proteins in the MMR pathway, allowing the removal of the mismatch and resynthesis of the resulting gap (reviewed in references 42 and 60).…”

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confidence: 99%

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

et al. 2010

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The ␤ clamp is an essential replication sliding clamp required for processive DNA synthesis. The ␤ clamp is also critical for several additional aspects of DNA metabolism, including DNA mismatch repair (MMR). The dnaN5 allele of Bacillus subtilis encodes a mutant form of ␤ clamp containing the G73R substitution. Cells with the dnaN5 allele are temperature sensitive for growth due to a defect in DNA replication at 49°C, and they show an increase in mutation frequency caused by a partial defect in MMR at permissive temperatures. We selected for intragenic suppressors of dnaN5 that rescued viability at 49°C to determine if the DNA replication defect could be separated from the MMR defect. We isolated three intragenic suppressors of dnaN5 that restored growth at the nonpermissive temperature while maintaining an increase in mutation frequency. All three dnaN alleles encoded the G73R substitution along with one of three novel missense mutations. The missense mutations isolated were S22P, S181G, and E346K. Of these, S181G and E346K are located near the hydrophobic cleft of the ␤ clamp, a common site occupied by proteins that bind the ␤ clamp. Using several methods, we show that the increase in mutation frequency resulting from each dnaN allele is linked to a defect in MMR. Moreover, we found that S181G and E346K allowed growth at elevated temperatures and did not have an appreciable effect on mutation frequency when separated from G73R. Thus, we found that specific residue changes in the B. subtilis ␤ clamp separate the role of the ␤ clamp in DNA replication from its role in MMR.Replication sliding clamps are essential cellular proteins imparting a spectacular degree of processivity to DNA polymerases during genome replication (24,(39)(40)(41). Encoded by the dnaN gene, the ␤ clamp is a highly conserved bacterial sliding clamp found in virtually all eubacterial species (reviewed in reference 7). The ␤ clamp is a head-to-tail, ringshaped homodimer that encircles double-stranded DNA (1, 39). In eukaryotes and archaea, the analog of the ␤ clamp is proliferating cell nuclear antigen (PCNA) (15,28,40,41). Eukaryotic PCNA is a ring-shaped homotrimer that also acts to encircle DNA, increasing the processivity of the replicative DNA polymerases (40,41). Although the primary structures of the ␤ clamp and PCNA are not conserved, the tertiary structures of these proteins are very similar, demonstrating structural conservation among bacterial, archaeal, and eukaryotic replication sliding clamps (28, 39-41; reviewed in reference 6).The function of the ␤ clamp is not limited to its well-defined role in genome replication. The Escherichia coli ␤ clamp binds Hda, which also binds the replication initiation protein DnaA, regulating the active form of DnaA complexed with ATP (19,37,43). This allows the ␤ clamp to regulate replication initiation through the amount of available DnaA-ATP. In Bacillus subtilis, the ␤ clamp binds YabA, a negative regulator of DNA replication initiation (12,29,52). It has also been suggested that the B. subt...

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“…We first tested a series of chromosomal markers by using strain NR13145 [ara thi ⌬(pro-lac) mutL::Tn10 trpE9777] and its ⌬dinB::kan derivative, strain NR13146. The strains are derivatives of KA796 (32) into which the trpE9777 (30,34), mutL::Tn10 (31), and ⌬dinB::kan (21) alleles were introduced by P1 transduction. The strains allow measurement of trpE9777 frameshift reversion as well as forward mutagenesis to rifampin resistance (Rif r ) and nalidixic acid resistance (Nal r ).…”

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confidence: 99%

“…The strains allow measurement of trpE9777 frameshift reversion as well as forward mutagenesis to rifampin resistance (Rif r ) and nalidixic acid resistance (Nal r ). (34). The data shown in Fig.…”

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Role of Escherichia coli DNA Polymerase IV in In Vivo Replication Fidelity

et al. 2004

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We have investigated whether DNA polymerase IV (Pol IV; the dinB gene product) contributes to the error rate of chromosomal DNA replication in Escherichia coli. We compared mutation frequencies in mismatch repair-defective strains that were either dinB positive or dinB deficient, using a series of mutational markers, including lac targets in both orientations on the chromosome. Virtually no contribution of Pol IV to the chromosomal mutation rate was observed. On the other hand, a significant effect of dinB was observed for reversion of a lac allele when the lac gene resided on an F(pro-lac) episome.Several mechanisms control the fidelity of the DNA replication process. These include correct base selection by the DNA polymerase, removal of base insertion errors by 3Ј-exonucleolytic proofreading, and correction by DNA mismatch repair (29). In Escherichia coli, base selection and proofreading are performed by the DNA polymerase III (Pol III) holoenzyme, the enzyme that replicates the bacterial chromosome. It is generally considered a highly accurate enzyme (29). Mismatch repair is performed by the mutHLS mismatch repair system (17). In combination, these three processes yield an error rate of 10 Ϫ9 to 10 Ϫ11 error per base pair replicated per cell division (6,29).In addition to Pol III, E. coli possesses four other DNA polymerases, Pol I, Pol II, Pol IV, and Pol V, whose precise functions are still being defined. Pol IV and Pol V belong to the recently described Y family of DNA polymerases

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The reversion of trp frameshift mutations in mut, polA, lig and dnaE mutant strains of Escherichia coli

Cited by 40 publications

References 22 publications

Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli : Analysis of the dnaX36 Mutator Mutant

Role of Accessory DNA Polymerases in DNA Replication in Escherichia coli : Analysis of the dnaX36 Mutator Mutant

Mutations in the Bacillus subtilis β Clamp That Separate Its Roles in DNA Replication from Mismatch Repair

Role of Escherichia coli DNA Polymerase IV in In Vivo Replication Fidelity

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