Cloning and deletion mapping of the recF dnaN region of the Escherichia coli chromosome

Ream, Lloyd W.; Clark, Alvin J.

doi:10.1016/0147-619x(83)90062-8

Cited by 14 publications

(8 citation statements)

References 33 publications

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“…The nucleotide sequence of a 1.325-kb Msp I-EcoRI fragment, which has been shown to contain a completely functional recF gene (ref. 22; unpublished results) is presented in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

“…pJC605 is a 7.9-kilobase (kb) plasmid derived by cloning a 3.6-kb fragment carrying recF, called cJC1, with pBR322 (22). pMAB4 is a 3.6-kb plasmid derived by cloning the 1.3-kb EcoRI-Pvu II recF+ fragment of cJC1 into the 2.3-kb EcoRI-Pvu II bla' fragment of pBR322.…”

Section: Methodsmentioning

confidence: 99%

“…pMAB4 is a 3.6-kb plasmid derived by cloning the 1.3-kb EcoRI-Pvu II recF+ fragment of cJC1 into the 2.3-kb EcoRI-Pvu II bla' fragment of pBR322. pJC655 is a 7.7-kb de(bla)300 ampicillin-sensitive (Amps) deletion mutant derivative of pJC605 (22). Tn3 mutant derivatives of pJC655 (12.7 kb) were isolated by conduction with the conjugative plasmid pJC753.…”

Section: Methodsmentioning

confidence: 99%

“…These effects are plausibly explained by hypothesizing that recF encodes an endonuclease (21) or a protein involved in DNA synthesis. Cloning of the recF region has been described (22), and in this paper we identify the recF gene product.…”

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confidence: 99%

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Molecular analysis of the recF gene of Escherichia coli.

Blanar¹,

Sandler²,

Armengod³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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We analyzed the nucleotide sequence of a 1.325-kilobase region of wild-type Escherichia coli containing a functional recF gene and six Tn3 mutations that inactivate recF. The analysis shows a potentially translatable reading frame of 1071 nucleotides, which is interrupted by all six insertions. A protein of 40.5 kilodaltons would result from translation of the open reading frame, and a radioactive band of protein of an apparent molecular weight of %40 kilodaltons was seen by the maxicell method using a recF+ plasmid. Putative truncated peptides were seen when two recF::Tn3 mutant plasmids were used. Differential expression of dnaN and recF from a common promoter was noted. recF332::Tn3 was transferred to the chromosome where, in hemizygous condition, it produced UV sensitivity indistinguishable from that produced by two presumed recF point mutations.The recF gene of Escherichia coli lies within a cluster of genes involved in DNA metabolism, the gene order being gyrB recF dnaN dnaA (1). gyrB has been shown to code for one of the subunits of DNA gyrase (2), dnaN determines the 13 subunit of DNA polymerase III holoenzyme (3), and dnaA encodes a 52-kilodalton (kDa) protein (4,5) involved with the initiation of replication of the Escherichia coli chromosome (6).The phenotype of a recF mutant indicates that recF also is involved in DNA metabolism and possibly with replication.

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“…The nucleotide sequence of a 1.325-kb Msp I-EcoRI fragment, which has been shown to contain a completely functional recF gene (ref. 22; unpublished results) is presented in Fig. 1.…”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular analysis of the recF gene of Escherichia coli.

Blanar¹,

Sandler²,

Armengod³

et al. 1984

Proc. Natl. Acad. Sci. U.S.A.

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“…Therefore, we hypothesized that the UV sensitivity of the dnaN159 strain would be epistatic with mutations that impair the RecFOR-mediated pathway of ssDNA gap repair, which is reported to utilize both Pol I and Pol III for gap filling (16,37). Since the recF allele is located immediately downstream of dnaN (32,33), we chose to construct isogenic E. coli strains bearing dnaN ϩ or dnaN159, together with either the recO ϩ , ⌬recO1504::Tn5 (20), recR ϩ , or ⌬recR252::Tn10ϳ9 (25) alleles, and subsequently compared their respective levels of sensitivity to UV irradiation. Consistent with our previous findings (38), the dnaN159 strain was ϳ10-fold more sensitive to UV irradiation than was the isogenic dnaN ϩ strain (Fig.…”

Section: Resultsmentioning

confidence: 99%

Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

et al. 2007

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The Escherichia coli dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that is impaired for interaction with the replicative DNA polymerase (Pol), Pol III. In addition, strains bearing the dnaN159 allele require functional Pol I for viability. We have utilized a combination of genetic and biochemical approaches to characterize the role(s) played by Pol I in the dnaN159 strain. Our findings indicate that elevated levels of Pol I partially suppress the temperature-sensitive growth phenotype of the dnaN159 strain.In addition, we demonstrate that the ␤ clamp stimulates the processivity of Pol I in vitro and that ␤159 is impaired for this activity. The reduced ability of ␤159 to stimulate Pol I in vitro correlates with our finding that single-stranded DNA (ssDNA) gap repair is impaired in the dnaN159 strain. Taken together, these results suggest that (i) the ␤ clamp-Pol I interaction may be important for proper Pol I function in vivo and (ii) in the absence of Pol I, ssDNA gaps may persist in the dnaN159 strain, leading to lethality of the dnaN159 ⌬polA strain.The Escherichia coli dnaN-encoded ␤-sliding clamp functions as a homodimer and is "loaded" onto primed DNA by the multisubunit DnaX clamp loader complex (5, 21). Once loaded, the ␤ clamp slides freely along duplex DNA and functions to tether a variety of different proteins involved in replication and repair to the DNA (4,6,7,9,19,23,24,26,28,(41)(42)(43). The dnaN159 allele encodes a mutant form of the ␤-sliding clamp (␤159) that bears two amino acid substitutions: G66E (glycine at position 66 replaced with glutamic acid) and G174A (14,35,38). The G174A substitution appears to impair the ability of a hydrophobic cleft in ␤ to interact with the eubacterial clamp-binding motif (QL[S/D]LF) that is conserved in most partner proteins reported to interact with the ␤ clamp (8, 38). E. coli strains bearing the dnaN159 allele display temperature-sensitive growth (14,35,38) and altered DNA polymerase (Pol) usage (29,(38)(39)(40). These phenotypes appear to result, at least in part, from impaired interactions of the mutant ␤159 clamp protein with the replicative DNA polymerase, Pol III (38). As a result of the impaired ␤159-Pol III interaction, E. coli strains bearing the dnaN159 allele display increased utilization of the three SOS-regulated DNA polymerases, Pol II (polB), Pol IV (dinB), and Pol V (umuDC) (29,(38)(39)(40). In contrast to these three Pols, which under certain conditions impede growth of the dnaN159 strain (29, 39, 40), presumably by impairing DNA replication, the catalytic DNA polymerase activity of Pol I (polA) is essential for viability of the dnaN159 strain (38). Importantly, Pol I function is not required by the isogenic dnaN ϩ strain (38), indicating that Pol I plays one or more essential roles in the dnaN159 strain. Since Pol I is a multifunctional protein that participates in DNA replication, as well as numerous DNA repair pathways, several possibilities exist, including Okazaki fragment maturation (21, 31) and single-stranded DNA ...

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Heterogeneity in the level of ampicillin resistance conferred by pBR322 derivatives with different DNA supercoiling

Aleixandre

Blanco

1987

Mol Gen Genet

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Cloning of an EcoRI restriction fragment, containing the 900 bp gamma-terminal sequence of transposon Tn1000, into pBR322, resulted in two plasmids, pICV63 and pICV64, which differed in the orientation of the cloned fragment within the replicon and in the level of ampicillin resistance conferred on the host cell. The DNAs of these plasmids differ in superhelicity and we suggest that a change in supercoiling of pICV63 DNA leads to this plasmid conferring resistance to only low levels of ampicillin, probably by reducing the expression of the bla gene. This hypothesis is supported by the fact that topA or supX mutations, which abolish topoisomerase I, reduce still further the level of resistance to ampicillin of pICV63-containing cells, whereas the gyrB226 compensatory mutation renders these cells more ampicillin resistant. Plasmid pICV63, therefore, enables mutant alleles of genes governing DNA topology to be recognized.

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Cloning and deletion mapping of the recF dnaN region of the Escherichia coli chromosome

Cited by 14 publications

References 33 publications

Molecular analysis of the recF gene of Escherichia coli.

Molecular analysis of the recF gene of Escherichia coli.

Role of Escherichia coli DNA Polymerase I in Conferring Viability upon the dnaN159 Mutant Strain

Heterogeneity in the level of ampicillin resistance conferred by pBR322 derivatives with different DNA supercoiling

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