Initiation of DNA Synthesis in Escherichia Coli

Tomizawa, Jun-ichi; Selzer, Gerald

doi:10.1146/annurev.bi.48.070179.005031

Cited by 103 publications

(37 citation statements)

References 82 publications

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“…To show the general applicability of the agarose gel retardation assay and examine the type of complexes formed, we used purified DNA fragments carrying sites previously shown to bind DnaA protein in vitro: the pUC origin (15), oriC (15), and the -y origin core (Sla). The involvement of DnaA in replication of pBR322 (40a) and oriC (16,17,24,48) has been described previously.…”

Section: Schaefer and Messer (40) (T/c)(t/c)(a/t/c)t(a/c)c(a/g) (A/cmentioning

confidence: 85%

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Levchenko

Filutowicz

1994

J Bacteriol

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Replication of the -y origin ofEscherichia coli plasmid R6K requires i protein, encoded by the R6Kpir gene, and many host factors, including DnaA protein. zr has dual roles, activating replication at low levels and inhibiting replication at high levels. The inhibitory function of w is counteracted by integration host factor and a specific sequence of the origin called the enhancer. This 106-bp DNA segment contains a binding site for DnaA protein (DnaA box 1). In this study, we mutated this site to determine if it was required for the enhancer's function. Using -y origin derivative plasmids with the DnaA box 1 altered or deleted, we show that this site is necessary to protect the origin against levels of wild-type wF protein that would otherwise inhibit replication. To show that the base substitutions in DnaA box 1 weakened the binding of DnaA, we developed a new application of the agarose gel retardation assay. This quick and easy assay has broad applicability, as shown in binding studies with DNA fragments carrying a different segment of the R6K origin, the chromosomal origin (oriC), or the pUC origin. The gel retardation assay suggests a stoichiometry of DnaA binding different from that deduced from other assays.Many replication origins have a bipartite organization, including an essential segment and an auxiliary segment which augments replication. Such enhancers have been discovered in organisms and systems as diverse as, for example, the bacteriophage fl (8, 23), the Staphylococcus aureus plasmid pT181 (21), and the yeast autonomously replicating sequence ARS121 (49). In each of these cases, the enhancer carries, or is believed to carry, a protein-binding site (20, 37a, 49, 50).The Escherichia coli plasmid R6K contains three origins of replication, a, y, and ,3 ( Fig. 1), which all require the initiator protein 7r, encoded by the R6K pir gene. All three origins require the essential 277-bp core segment, which contains repeated binding sites (iterons) for TT (14,19,33); each origin also needs a unique cis-acting DNA segment in addition to the core (28,29,36,41,42,44). When the flanking ot and P sequences are removed, the remaining y origin can replicate autonomously (18,43,46) if thepir gene is provided in cis or in trans (22,29). The y origin responds to the intracellular concentration of uz protein, and it contains the most critical information required for replication (12,28). Therefore, we use the y origin system (for the most recent review, see reference 10) as the simplest model derived from R6K to study the mechanisms regulating initiation of replication of an iteron-containing origin.The -y origin consists of two modules, the essential 277-bp core segment and the adjoining 106-bp enhancer (51a) (Fig. 1). The enhancer is not required for the a or ,B origin (36, 41, 42, 44). The isolated core is able to replicate if the level of wild-type (wt) wT is decreased (Sla), a situation which reduces the stringency of replication control (12). At the normal level of wt 7r (4,000 to 10,000 IT (9,11,25), the p...

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Section: Schaefer and Messer (40) (T/c)(t/c)(a/t/c)t(a/c)c(a/g) (A/cmentioning

confidence: 85%

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Levchenko

Filutowicz

1994

J Bacteriol

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“…The protein product of the bacterial dnaA gene is required for in vivo initiation of DNA replication at oriC in E. coli (45). A number of experimental observations have led to speculation that DnaA is involved in coupling DNA initiation to the oriC/mass ratio of a cell (2).…”

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confidence: 99%

Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion

1994

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The Escherichia coli nrd operon contains the genes encoding the two subunits of ribonucleoside diphosphate reductase. The regulation of the nrd operon has been observed to be very complex. The specific binding of two proteins to the nrd regulatory region and expression of mutant nrd-lac fusions that do not bind these proteins are described. A partially purified protein from an E. coli cell extract was previously shown to bind to the promoter region and to regulate transcription of the nrd operon (C. K. Tuggle and J. A. Fuchs, J. Bacteriol. 172:1711-1718, 1990 starved cells. The protein extract from a thymine-starved culture protected several sites in the promoter region from DNase I digestion (47). Here, we report the identity of this protein as the E. coli factor for inversion stimulation (Fis).Fis is an 11.2-kDa protein initially identified by its ability to stimulate Hin-, Gin-, and Cin-mediated DNA inversion (18,23,25). The involvement of Fis in other site-specific recombinations, including excision and integration recombination reactions of bacteriophage X DNA, was also demonstrated (3,4,44). Fis has also been shown to be a transcriptional activator at rRNA and tRNA promoters in E. coli (6,38). In vitro footprinting experiments show protection of specific sites in the E. coli oriC sequence by Fis (17), and examination of DNA synthesis in fis gyrB double mutants indicates a synergistic involvement of Fis and DNA gyrase in the initiation of DNA replication at oriC (14).The fis gene has been cloned, sequenced, and mapped to 72 min on the E. coli chromosome (23,26). The gene encodes an 11,239-Da, 98-amino-acid polypeptide. The fis operon promoter has been located 1 kb upstream of the fis start codon (36), and Fis synthesis is autoregulated. Transcription of fis is controlled as part of the stringent response. A degenerate consensus binding site for Fis has been proposed: (G/T) nnYRnn(A/T)nnYRnn(C/A) (where Y is a pyrimidine, R is a purine, and n is any nucleotide) (22). However, local DNA topology and the ability of the DNA duplex to bend, accommodating the helix-turn-helix DNA-binding domains of the Fis dimer, appear to be more important in determining Fis binding at any given site (51) than the sequence per se. In activating transcription of stable RNA operons in E. coli, binding of Fis to upstream activating sequences lowers the dissociation constant of the RNA polymerase-promoter complex (34).

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“…DNA synthesis in fis and gyrB double mutants indicates a synergistic involvement of Fis and DNA gyrase in the initiation of DNA replication at oriC (19). DnaA protein is required to initiate DNA replication at oriC in E. coli (49) and to coordinate the synchrony of chromosome replication (42). DNA footprinting and site-directed mutations indicate that the DnaA protein specifically binds to two DnaA boxes located between -35 and -55 relative to the start of nrd transcription (3).…”

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Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

et al. 1994

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The expression of the nrd operon encoding ribonucleotide reductase in Escherichia coli has been shown to be cell cycle regulated. To identity the cis-acting elements required for the cell cycle regulation of the nrd promoter, different 5' deletions as well as site-directed mutations were translationally fused to a lacZ reporter gene. The expression of 0-galactosidase from these nrd-lacZ fusions in single-copy plasmids was Ribonucleotide reductase (RR) catalyzes the enzymatic reduction of ribonucleotides to deoxyribonucleotides and is the first enzyme in the pathway unique to DNA replication in Escherichia coli. The two nonidentical subunits of RR encoded by the nrdA and nrdB genes (20) are transcribed as a 3.2-kb polycistronic mRNA (23). The nrd operon has been sequenced, including approximately 3 kb of the upstream flanking sequence (8). The nrd promoter region was analyzed, and the operon's start of transcription was mapped at 110 bp upstream of the nrdA AUG start codon (50). Conditions that result in an increased mass/DNA ratio result in increased levels of RR (17). The synthesis of RR mRNA increases when DNA synthesis is specifically inhibited (17,18,23). Protein synthesis is necessary for both the initial induction and the continued increase of nrd mRNA synthesis during thymine starvation. Studies with cultures synchronized by either repeated phosphate starvation or sucrose gradient centrifugation demonstrated that the expression of the nrd operon in E. coli is cell cycle regulated (48). nrd mRNA synthesis increased at a time corresponding to the initiation of DNA replication. This type of regulation is similar to that observed in eukaryotic organisms (2,(13)(14)(15)(16)35 Flow cytometry is widely used in the analysis and sorting of viable heterogeneous cell populations. In the past decade, flow cytometry has been used for measuring cell cycle-related properties of a variety of mammalian cell cultures and has had major impact on cell cycle studies of mammalian cells. Recently, flow cytometry techniques have been successfully employed in cell cycle studies in the yeast Saccharomyces cerevisiae (11,12,43) and in DNA replication studies in the bacterium E. coli (6,38,41,(44)(45)(46)(47)52).In this study, we have used a series of nrd-lacZ fusion plasmid constructs to determine the cis-acting elements of the nrd regulatory region that are required for cell cycle-regulated nrd expression. Two different experimental approaches were used. In synchronized cultures, DNA replication and lac mRNA produced from the nrd-lac fusion located on singlecopy-number vectors as well as the nrd mRNA produced from the chromosomal gene were analyzed. Alteration in expression caused by mutations in the nrd promoter of the nrd-lac fusion on the vector could be compared with the chromosomal nrd promoter by comparing the lac mRNA and the nrd mRNA. In a second experimental approach, we used flow cytometry analysis with a combined immunofluorescent staining of n-galactosidase and a DNA-specific stain to quantitate 3-galacto-2415

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Initiation of DNA Synthesis in Escherichia Coli

Cited by 103 publications

References 82 publications

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Binding of DnaA protein to a replication enhancer counteracts the inhibition of plasmid R6K gamma origin replication mediated by elevated levels of R6K pi protein

Escherichia coli Fis and DnaA proteins bind specifically to the nrd promoter region and affect expression of an nrd-lac fusion

Cell cycle regulation of the Escherichia coli nrd operon: requirement for a cis-acting upstream AT-rich sequence

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