scholarly journals Quantitative analysis of nucleotide modulation of DNA binding by DnaC protein of Escherichia coli

2004 ◽  
Vol 379 (3) ◽  
pp. 553-562 ◽  
Author(s):  
Subhasis B. BISWAS ◽  
Stephen FLOWERS ◽  
Esther E. BISWAS-FISS
Keyword(s):  
Escherichia Coli ◽  
Quantitative Analysis ◽  
Dna Replication ◽  
Dna Binding ◽  
Binding Affinity ◽  
Replication Fork ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Chromosomal Dna ◽  
Dnac Protein

In this study, we have presented the first report of Escherichia coli DnaC protein binding to ssDNA (single stranded DNA) in an apparent hexameric form. DnaC protein transfers DnaB helicase onto a nascent chromosomal DNA replication fork at oriC, the origin of E. coli DNA replication. In eukaryotes, Cdc6 protein may play a similar role in the DNA helicase loading in the replication fork during replication initiation at the origin. We have analysed the DNA-binding properties of DnaC protein and a quantitative analysis of the nucleotide regulation of DnaC–DNA and DnaC–DnaB interactions using fluorescence anisotropy and affinity sensor analysis. DnaC protein bound to ssDNA with low to moderate affinity and the affinity was strictly modulated by nucleotides. DnaC bound ssDNA in the complete absence of nucleotides. The DNA-binding affinity was significantly increased in the presence of ATP, but not ATP[S]. In the presence of ADP, the binding affinity decreased approximately fifty-fold. Both anisotropy and biosensor analyses demonstrated that with DnaC protein, ATP facilitated ssDNA binding, whereas ADP facilitated its dissociation from ssDNA, which is a characteristic of an ATP/ADP switch. Both ssDNA and nucleotides modulate DnaB6•DnaC6 complex formation, which has significant implications in DnaC protein function. Based on the thermodynamic data provided in this study, we have proposed a mechanism of DnaB loading on to ssDNA by DnaC protein.

scholarly journals DpiA Binding to the Replication Origin of Escherichia coli Plasmids and Chromosomes Destabilizes Plasmid Inheritance and Induces the Bacterial SOS Response

2003 ◽  
Vol 185 (20) ◽  
pp. 6025-6031 ◽  
Author(s):  
Christine Miller ◽  
Hanne Ingmer ◽  
Line Elnif Thomsen ◽  
Kirsten Skarstad ◽  
Stanley N. Cohen
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Origin ◽  
Sos Response ◽  
Dna Helicase ◽  
Replication Initiation ◽  
Chromosomal Dna ◽  
E Coli ◽  
Dna Replication Initiation ◽  
Helicase Dnab

ABSTRACT The dpiA and dpiB genes of Escherichia coli, which are orthologs of genes that regulate citrate uptake and utilization in Klebsiella pneumoniae, comprise a two-component signal transduction system that can modulate the replication of and destabilize the inheritance of pSC101 and certain other plasmids. Here we show that perturbed replication and inheritance result from binding of the effector protein DpiA to A+T-rich replication origin sequences that resemble those in the K. pneumoniae promoter region targeted by the DpiA ortholog, CitB. Consistent with its ability to bind to A+T-rich origin sequences, overproduction of DpiA induced the SOS response in E. coli, suggesting that chromosomal DNA replication is affected. Bacteria that overexpressed DpiA showed an increased amount of DNA per cell and increased cell size—both also characteristic of the SOS response. Concurrent overexpression of the DNA replication initiation protein, DnaA, or the DNA helicase, DnaB—both of which act at A+T-rich replication origin sequences in the E. coli chromosome and DpiA-targeted plasmids—reversed SOS induction as well as plasmid destabilization by DpiA. Our finding that physical and functional interactions between DpiA and sites of replication initiation modulate DNA replication and plasmid inheritance suggests a mechanism by which environmental stimuli transmitted by these gene products can regulate chromosomal and plasmid dynamics.

Overlap of replication rounds disturbs the progression of replicating forks in a ribonucleotide reductase mutant of Escherichia coli

Microbiology ◽  
2011 ◽  
Vol 157 (7) ◽  
pp. 1955-1967 ◽  
Author(s):  
Israel Salguero ◽  
Elena López Acedo ◽  
Elena C. Guzmán
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Ribonucleotide Reductase ◽  
Replication Fork ◽  
Rna Synthesis ◽  
Restrictive Temperature ◽  
Protein Synthesis Inhibition ◽  
Chromosomal Dna ◽  
Dnaa Protein ◽  
Density Results

Ribonucleotide reductase (RNR) is the only enzyme specifically required for the synthesis of deoxyribonucleotides (dNTPs). Surprisingly, Escherichia coli cells carrying the nrdA101 allele, which codes for a thermosensitive RNR101, are able to replicate entire chromosomes at 42 °C under RNA or protein synthesis inhibition. Here we show that the RNR101 protein is unstable at 42 °C and that its degradation under restrictive conditions is prevented by the presence of rifampicin. Nevertheless, the mere stability of the RNR protein at 42 °C cannot explain the completion of chromosomal DNA replication in the nrdA101 mutant. We found that inactivation of the DnaA protein by using several dnaAts alleles allows complete chromosome replication in the absence of rifampicin and suppresses the nucleoid segregation and cell division defects observed in the nrdA101 mutant at 42 °C. As both inactivation of the DnaA protein and inhibition of RNA synthesis block the occurrence of new DNA initiations, the consequent decrease in the number of forks per chromosome could be related to those effects. In support of this notion, we found that avoiding multifork replication rounds by the presence of moderate extra copies of datA sequence increases the relative amount of DNA synthesis of the nrdA101 mutant at 42 °C. We propose that a lower replication fork density results in an improvement of the progression of DNA replication, allowing replication of the entire chromosome at the restrictive temperature. The mechanism related to this effect is also discussed.

scholarly journals lon Incompatibility Associated with Mutations Causing SOS Induction: Null uvrD Alleles Induce an SOS Response in Escherichia coli

2000 ◽  
Vol 182 (11) ◽  
pp. 3151-3157 ◽  
Author(s):  
L. SaiSree ◽  
Manjula Reddy ◽  
J. Gowrishankar
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Mismatch Repair ◽  
Replication Fork ◽  
Excision Repair ◽  
Dna Helicase ◽  
Lon Protease ◽  
Sos Induction ◽  
Nucleotide Excision ◽  
Lagging Strand

ABSTRACT The uvrD gene in Escherichia coli encodes a 720-amino-acid 3′-5′ DNA helicase which, although nonessential for viability, is required for methyl-directed mismatch repair and nucleotide excision repair and furthermore is believed to participate in recombination and DNA replication. We have shown in this study that null mutations in uvrD are incompatible withlon, the incompatibility being a consequence of the chronic induction of SOS in uvrD strains and the resultant accumulation of the cell septation inhibitor SulA (which is a normal target for degradation by Lon protease). uvrD-lonincompatibility was suppressed by sulA,lexA3(Ind−), or recA (Def) mutations. Other mutations, such as priA, dam,polA, and dnaQ (mutD) mutations, which lead to persistent SOS induction, were also lonincompatible. SOS induction was not observed in uvrC andmutH (or mutS) mutants defective, respectively, in excision repair and mismatch repair. Nor wasuvrD-mediated SOS induction abolished by mutations in genes that affect mismatch repair (mutH), excision repair (uvrC), or recombination (recB andrecF). These data suggest that SOS induction inuvrD mutants is not a consequence of defects in these three pathways. We propose that the UvrD helicase participates in DNA replication to unwind secondary structures on the lagging strand immediately behind the progressing replication fork, and that it is the absence of this function which contributes to SOS induction inuvrD strains.

scholarly journals The Absence of (p)ppGpp Renders Initiation of Escherichia coli Chromosomal DNA Synthesis Independent of Growth Rates

mBio ◽  
2020 ◽  
Vol 11 (2) ◽  
Author(s):  
Llorenç Fernández-Coll ◽  
Monika Maciag-Dorszynska ◽  
Krishma Tailor ◽  
Stephen Vadia ◽  
Petra Anne Levin ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Growth Rate ◽  
Dna Replication ◽  
Dna Synthesis ◽  
Growth Rates ◽  
Exponential Growth ◽  
Replication Initiation ◽  
Fast Growth ◽  
Chromosomal Dna ◽  
Wide Range

ABSTRACT The initiation of Escherichia coli chromosomal DNA replication starts with the oligomerization of the DnaA protein at repeat sequences within the origin (ori) region. The amount of ori DNA per cell directly correlates with the growth rate. During fast growth, the cell generation time is shorter than the time required for complete DNA replication; therefore, overlapping rounds of chromosome replication are required. Under these circumstances, the ori region DNA abundance exceeds the DNA abundance in the termination (ter) region. Here, high ori/ter ratios are found to persist in (p)ppGpp-deficient [(p)ppGpp0] cells over a wide range of balanced exponential growth rates determined by medium composition. Evidently, (p)ppGpp is necessary to maintain the usual correlation of slow DNA replication initiation with a low growth rate. Conversely, ori/ter ratios are lowered when cell growth is slowed by incrementally increasing even low constitutive basal levels of (p)ppGpp without stress, as if (p)ppGpp alone is sufficient for this response. There are several previous reports of (p)ppGpp inhibition of chromosomal DNA synthesis initiation that occurs with very high levels of (p)ppGpp that stop growth, as during the stringent starvation response or during serine hydroxamate treatment. This work suggests that low physiological levels of (p)ppGpp have significant functions in growing cells without stress through a mechanism involving negative supercoiling, which is likely mediated by (p)ppGpp regulation of DNA gyrase. IMPORTANCE Bacterial cells regulate their own chromosomal DNA synthesis and cell division depending on the growth conditions, producing more DNA when growing in nutritionally rich media than in poor media (i.e., human gut versus water reservoir). The accumulation of the nucleotide analog (p)ppGpp is usually viewed as serving to warn cells of impending peril due to otherwise lethal sources of stress, which stops growth and inhibits DNA, RNA, and protein synthesis. This work importantly finds that small physiological changes in (p)ppGpp basal levels associated with slow balanced exponential growth incrementally inhibit the intricate process of initiation of chromosomal DNA synthesis. Without (p)ppGpp, initiations mimic the high rates present during fast growth. Here, we report that the effect of (p)ppGpp may be due to the regulation of the expression of gyrase, an important enzyme for the replication of DNA that is a current target of several antibiotics.

scholarly journals Delineation of the Ancestral Tus-Dependent Replication Fork Trap

2021 ◽  
Author(s):  
Casey Toft ◽  
Morgane Moreau ◽  
Jiri Perutka ◽  
Savitri Mandapati ◽  
Peter Enyeart ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Replication Fork ◽  
Wide Distribution ◽  
Replication Forks ◽  
Genome Wide ◽  
Replication Fork Arrest

In Escherichia coli, DNA replication termination is orchestrated by two clusters of Ter sites forming a DNA replication fork trap when bound by Tus proteins. The formation of a ‘locked’ Tus-Ter complex is essential for halting incoming DNA replication forks. However, the absence of replication fork arrest at some Ter sites raised questions about their significance. In this study, we examined the genome-wide distribution of Tus and found that only the six innermost Ter sites (TerA-E and G) were significantly bound by Tus. We also found that a single ectopic insertion of TerB in its non-permissive orientation could not be achieved, advocating against a need for ‘back-up’ Ter sites. Finally, examination of the genomes of a variety of Enterobacterales revealed a new replication fork trap architecture mostly found outside the Enterobacteriaceae family. Taken together, our data enabled the delineation of a narrow ancestral Tus-dependent DNA replication fork trap consisting of only two Ter sites.

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