DNA replication in Escherichia coli gyrB(Ts) mutants analysed by flow cytometry

Freiesleben, Ulrik von; Rasmussen, Knud V.

doi:10.1016/0923-2508(91)90034-8

Cited by 11 publications

(8 citation statements)

References 18 publications

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“…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).…”

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confidence: 99%

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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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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“…Transcriptional activation of gidA , which is adjacent to oriC in E. coli , can alter topology at the origin by introducing negative supercoiling within the region (Asai et al ., ; Bogan and Helmstetter, ). Furthermore, decreased supercoiling was shown to lead to asynchronous replication initiation (Von Freiesleben and Rasmussen, ). Additionally, initiation phenotypes observed in the temperature sensitive alleles of dnaA could be suppressed by mutations in topA (Topoisomerase I) (Louarn et al ., ).…”

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confidence: 99%

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

Samadpour

Merrikh

2018

Molecular Microbiology

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In bacteria, initiation of DNA replication requires the DnaA protein. Regulation of DnaA association and activity at the origin of replication, oriC, is the predominant mechanism of replication initiation control. One key feature known to be generally important for replication is DNA topology. Although there have been some suggestions that topology may impact replication initiation, whether this mechanism regulates DnaA-mediated replication initiation is unclear. We found that the essential topoisomerase, DNA gyrase, is required for both proper binding of DnaA to oriC as well as control of initiation frequency in Bacillus subtilis. Furthermore, we found that the regulatory activity of gyrase in initiation is specific to DnaA and oriC. Cells initiating replication from a DnaA-independent origin, oriN, are largely resistant to gyrase inhibition by novobiocin, even at concentrations that compromise survival by up to four orders of magnitude in oriC cells. Furthermore, inhibition of gyrase does not impact initiation frequency in oriN cells. Additionally, deletion or overexpression of the DnaA regulator, YabA, significantly modulates sensitivity to gyrase inhibition, but only in oriC and not oriN cells. We propose that gyrase is a negative regulator of DnaA-dependent replication initiation from oriC, and that this regulatory mechanism is required for cell survival.

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“…Increased supercoiling, as is seen in strains with mutations in topA encoding Topoisomerase I, suppresses the temperature sensitivity of the dnaA46 (ts) allele, which normally under-initiates (157). Decreased supercoiling also causes asynchronous initiations from oriC (246, 247), as well as adversely affecting minichromosome maintenance (150). These findings suggest a direct relationship between DNA supercoiling and initiation efficiency, most likely by modulation of the amount of DnaA required to unwind oriC .…”

Section: Factors Involved In Initiation Of Chromosome Replicationmentioning

confidence: 99%

Initiation of DNA Replication

Leonard

Grimwade

2010

EcoSal Plus

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In recent years it has become clear that complex regulatory circuits control the initiation step of DNA replication by directing the assembly of a multicomponent molecular machine (the orisome) that separates DNA strands and loads replicative helicase at oriC , the unique chromosomal origin of replication. This chapter discusses recent efforts to understand the regulated protein-DNA interactions that are responsible for properly timed initiation of chromosome replication. It reviews information about newly identified nucleotide sequence features within Escherichia coli oriC and the new structural and biochemical attributes of the bacterial initiator protein DnaA. It also discusses the coordinated mechanisms that prevent improperly timed DNA replication. Identification of the genes that encoded the initiators came from studies on temperature-sensitive, conditional-lethal mutants of E. coli , in which two DNA replication-defective phenotypes, "immediate stop" mutants and "delayed stop" mutants, were identified. The kinetics of the delayed stop mutants suggested that the defective gene products were required specifically for the initiation step of DNA synthesis, and subsequently, two genes, dnaA and dnaC , were identified. The DnaA protein is the bacterial initiator, and in E. coli , the DnaC protein is required to load replicative helicase. Regulation of DnaA accessibility to oriC , the ordered assembly and disassembly of a multi-DnaA complex at oriC , and the means by which DnaA unwinds oriC remain important questions to be answered and the chapter discusses the current state of knowledge on these topics.

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DNA replication in Escherichia coli gyrB(Ts) mutants analysed by flow cytometry

Cited by 11 publications

References 18 publications

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

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

DNA gyrase activity regulates DnaA‐dependent replication initiation in Bacillus subtilis

Initiation of DNA Replication

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