Genetic and physical mapping of recF in Escherichia coli K-12

Ream, Lloyd W.; Margossian, Linda; Clark, Alvin J.; Hansen, Flemming; Meyenburg, Kaspar von

doi:10.1007/bf00267359

Cited by 53 publications

(26 citation statements)

References 29 publications

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“…In addition to its initiation role, DnaA represses the expression of many genes at the transcriptional level by binding (79). The dnaA gene itself, which forms an operon with other genes involved in DNA replication and metabolism, dnaN and recF (57) (Fig. 3), is autoregulated.…”

Section: Discussionmentioning

confidence: 99%

Mutations in Escherichia coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids

1995

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Extragenic suppressor mutations which had the ability to suppress a dnaX2016(Ts) DNA polymerization defect and which concomitantly caused cold sensitivity have been characterized within the dnaA initiation gene. When these alleles (designated Cs, Sx) were moved into dnaX ؉ strains, the new mutants became cold sensitive and phenotypically were initiation defective at 20؇C (J. R. Walker, J. A. Ramsey, and W. G. Haldenwang, Proc. Natl. Acad. Sci. USA 79:3340-3344, 1982). Detailed localization by marker rescue and DNA sequencing are reported here. One mutation changed codon 213 from Ala to Asp, the second changed Arg-432 to Leu, and the third changed codon 435 from Thr to Lys. It is striking that two of the three spontaneous mutations occurred in codons 432 and 435; these codons are within a very highly conserved, 12-residue region (K. Skarstad and E. Boye, Biochim. Biophys. Acta 1217:111-130, 1994; W. Messer and C. Weigel, submitted for publication) which must be critical for one of the DnaA activities. The dominance of wild-type and mutant alleles in both initiation and suppression activities was studied. First, in initiation function, the wild-type allele was dominant over the Cs, Sx alleles, and this dominance was independent of location. That is, the dnaA ؉ allele restored growth to dnaA(Cs, Sx) strains at 20؇C independently of which allele was present on the plasmid. The dnaA(Cs, Sx) alleles provided initiator function at 39؇C and were dominant in a dnaA(Ts) host at that temperature. On the other hand, suppression was dominant when the suppressor allele was chromosomal but recessive when it was plasmid borne. Furthermore, suppression was not observed when the suppressor allele was present on a plasmid and the chromosomal dnaA was a null allele. These data suggest that the suppressor allele must be integrated into the chromosome, perhaps at the normal dnaA location. Suppression by dnaA(Cs, Sx) did not require initiation at oriC; it was observed in strains deleted of oriC and which initiated at an integrated plasmid origin.The Escherichia coli dnaX gene encodes the and ␥ subunits of DNA polymerase III holoenzyme (20,38,51,86). , the larger protein (71.1 kDa), results from translation of the complete 643-codon message. ␥, the shorter product (47.5 kDa), results from a programmed Ϫ1 ribosomal frameshift over messenger codons 428 to 430 (10, 21, 72, 73, 75). This shift results in the translation of one unique amino acid, as residue 431, which is followed by a stop codon. The net result is that and ␥ are identical over the N-terminal 430 residues. The dnaX gene was defined by two temperature-sensitive (Ts) mutations, dnaX2016(Ts) (19) and dnaX36(Ts) (32). The dnaX2016(Ts) mutation changes codon 118 from glycine (GGT) to aspartate (GAT) and affects both and ␥ (7). Shifting the dnaX2016(Ts) mutant from the permissive temperature of 30ЊC to 42ЊC causes an immediate stop in DNA synthesis, as expected for a defect in polymerization, and growth gradually ceases (16). The dnaX36(Ts) mutation changes codon 601 from glutamat...

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Section: Discussionmentioning

confidence: 99%

Mutations in Escherichia coli dnaA which suppress a dnaX(Ts) polymerization mutation and are dominant when located in the chromosomal allele and recessive on plasmids

1995

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Section: Methodsmentioning

confidence: 99%

“…Bacterial cells were grown in Luria-Bertani (LB) medium at 22°C, which is the permissive temperature for AZ5372. QT186 (strain JC12334 [15]) has the tna-300::Tn10 marker, which is linked with the gyrB gene.Isolation of mutants that suppress novobiocin hypersensitivity of a mukB null mutation. Ten independent single colonies of strain AZ5372 grown on LB agar plates at 22°C were inoculated into LB liquid medium, and the cultures were grown to saturation at 22°C.…”

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confidence: 99%

Mutants Suppressing Novobiocin Hypersensitivity of amukBNull Mutation

Adachi

Hiraga

2003

J Bacteriol

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The mukB gene is essential for the partitioning of sister chromosomes in Escherichia coli. A mukB null mutant is hypersensitive to the DNA gyrase inhibitor novobiocin. In this work, we isolated mutants suppressing the novobiocin hypersensitivity of the mukB null mutation. All suppressor mutations are localized in or near the gyrB gene, and the four tested clones have an amino acid substitution in the DNA gyrase beta subunit. We found that in the mukB mutant, the process of sister chromosome segregation is strikingly hypersensitive to novobiocin; however, the effect of novobiocin on growth, which was measured by culture turbidity, is the same as that of the wild-type strain.The smtA-mukF-mukE-mukB operon (22) is located at the 21-min map position of the Escherichia coli chromosome. Deletion mutants of each muk gene show common phenotypes, such as frequent production of anucleate cells upon cell division, chromosome guillotining (cutting) by septum closure, and temperature-sensitive growth; however, they are nearly normal in chromosome replication, homologous recombination, mutation frequency, and sensitivity to UV irradiation (10, 11, 22; for a review, see reference 4). MukF, MukE, and MukB form a large complex (22). MukB is a member of the SMC superfamily (9). A MukB-green fluorescent protein fusion protein is localized as one, two, or four foci at regular cellular positions in the presence of both MukF and MukE (12). To examine the function of the MukB protein, many suppressor mutants of mukB mutations have been analyzed (for a review, see reference 4). Mutations of the topA gene encoding topoisomerase I suppress temperature-sensitive growth and anucleate-cell production (16). It has been demonstrated in synchronized cultures that replicated sister chromosomes are associated with one another for substantial times (6, 18). In contrast, results in randomly growing cultures in enriched medium appear to be consistent with the model in which replicated sister copies of oriC separate immediately after replication (8). However, it is hard to determine which model is right from the results of such random cultures, because the actual time of initiation of chromosome replication is not clear in this type of experiment.A mukB null mutant is hypersensitive to novobiocin, an inhibitor (20). Deletion mutants of each of the mukB, mukF, and mukE genes and a deletion mutant lacking all three muk genes are hypersensitive to novobiocin (14). Weitao et al. (20) showed that a seqA null mutation suppressed temperaturesensitive growth, anucleate-cell production, and novobiocin hypersensitivity in the mukB null mutation. Inconsistently, Onogi et al. (14) reported that a seqA or dam null mutation partially suppressed temperature-sensitive growth but failed to suppress the anucleate-cell production and novobiocin hypersensitivity of these muk null mutants.It is not yet clear what the mechanism of the novobiocin hypersensitivity of these muk mutants is. What is the target protein that is hypersensitive to novobiocin in these muk null...

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“…In the case of the RecF and RecR proteins, it is tempting to speculate about the possibility of a direct interaction with the replication proteins. The recF and recR genes are both found in operons that include the genes for some subunits of DNA polymerase III and other replication functions (Ream et al 1980;Flower & McHenry 1991;Perez-Roger et al 1991)-perhaps a coincidence of evolution and perhaps not.…”

Section: Quantifying Step 1 Under Normal Growth Conditionsmentioning

confidence: 99%