scholarly journals Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

2019 ◽  
Author(s):  
Reshma T Veetil ◽  
Nitish Malhotra ◽  
Akshara Dubey ◽  
Aswin Sai Narain Seshasayee
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Replication Initiation ◽  
Evolutionary Strategies ◽  
Rrna Genes ◽  
Sequence Motifs ◽  
Loop Formation ◽  
Laboratory Evolution ◽  
E Coli ◽  
Stable Dna Replication

AbstractThe bacterium E. coli can initiate replication in the absence of the replication initiator protein DnaA and / or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites, and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4-0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop forming regions; and includes a site containing sequence motifs that favour R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion however increases the possibility of conflicts at essential mRNA genes, which would utilise only a miniscule fraction of RNA polymerase molecules most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question.ImportanceThe bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures, and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics, and show that selection favours cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimised. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance against antibiotics that target initiation of DNA replication.

scholarly journals Laboratory Evolution Experiments Help Identify a Predominant Region of Constitutive Stable DNA Replication Initiation

mSphere ◽  
2020 ◽  
Vol 5 (1) ◽  
Author(s):  
Reshma T. Veetil ◽  
Nitish Malhotra ◽  
Akshara Dubey ◽  
Aswin Sai Narain Seshasayee
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Replication Initiation ◽  
Evolutionary Strategies ◽  
Rrna Genes ◽  
Loop Formation ◽  
Laboratory Evolution ◽  
Content Type ◽  
E Coli ◽  
Stable Dna Replication

ABSTRACT The bacterium Escherichia coli can initiate replication in the absence of the replication initiator protein DnaA and/or the canonical origin of replication oriC in a ΔrnhA background. This phenomenon, which can be primed by R-loops, is called constitutive stable DNA replication (cSDR). Whether DNA replication during cSDR initiates in a stochastic manner through the length of the chromosome or at specific sites and how E. coli can find adaptations to loss of fitness caused by cSDR remain inadequately answered. We use laboratory evolution experiments of ΔrnhA-ΔdnaA strains followed by deep sequencing to show that DNA replication preferentially initiates within a broad region located ∼0.4 to 0.7 Mb clockwise of oriC. This region includes many bisulfite-sensitive sites, which have been previously defined as R-loop-forming regions, and includes a site containing sequence motifs that favor R-loop formation. Initiation from this region would result in head-on replication-transcription conflicts at rRNA loci. Inversions of these rRNA loci, which can partly resolve these conflicts, help the bacterium suppress the fitness defects of cSDR. These inversions partially restore the gene expression changes brought about by cSDR. The inversion, however, increases the possibility of conflicts at essential mRNA genes, which would utilize only a minuscule fraction of RNA polymerase molecules, most of which transcribe rRNA genes. Whether subsequent adaptive strategies would attempt to resolve these conflicts remains an open question. IMPORTANCE The bacterium E. coli can replicate its DNA even in the absence of the molecules that are required for canonical replication initiation. This often requires the formation of RNA-DNA hybrid structures and is referred to as constitutive stable DNA replication (cSDR). Where on the chromosome does cSDR initiate? We answer this question using laboratory evolution experiments and genomics and show that selection favors cSDR initiation predominantly at a region ∼0.6 Mb clockwise of oriC. Initiation from this site will result in more head-on collisions of DNA polymerase with RNA polymerase operating on rRNA loci. The bacterium adapts to this problem by inverting a region of the genome including several rRNA loci such that head-on collisions between the two polymerases are minimized. Understanding such evolutionary strategies in the context of cSDR can provide insights into the potential causes of resistance to antibiotics that target initiation of DNA replication.

scholarly journals Evolution of DNA Replication Origin Specification and Gene Silencing Mechanisms

2020 ◽  
Author(s):  
Y. Hu ◽  
A. Tareen ◽  
Y-J. Sheu ◽  
W. T. Ireland ◽  
C. Speck ◽  
...  
Keyword(s):  
Dna Replication ◽  
Gene Silencing ◽  
Origin Recognition Complex ◽  
Replication Initiation ◽  
Separate Analysis ◽  
Genome Replication ◽  
Sequence Specificity ◽  
Sequence Motifs ◽  
Origin Firing ◽  
Α Helix

AbstractDNA replication in eukaryotic cells initiates from chromosomal locations, called replication origins, that bind the Origin Recognition Complex (ORC) prior to S phase. Origin establishment is guided by well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. At present, the mechanistic and evolutionary reasons for this difference are unclear. A 3.9 Å structure of S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed, among other things, that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove1. We show that this Orc4 α-helix mediates the sequence-specificity of origins in S. cerevisiae. Specifically, mutations were identified within this α-helix that alter the sequence-dependent activity of individual origins as well as change global genomic origin firing patterns. This was accomplished using a massively parallel origin selection assay analyzed using a custom mutual-information-based modeling approach and a separate analysis of whole-genome replication profiling and statistics. Interestingly, the sequence specificity of DNA replication initiation, as mediated by the Orc4 α-helix, has evolved in close conjunction with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference.

Initiation and termination of DNA replication in human rRNA genes

1993 ◽  
Vol 13 (10) ◽  
pp. 6600-6613
Author(s):  
R D Little ◽  
T H Platt ◽  
C L Schildkraut
Keyword(s):  
Dna Replication ◽  
Ribosomal Dna ◽  
Regulatory Elements ◽  
Transcription Unit ◽  
Replication Initiation ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Replication Forks ◽  
Nontranscribed Spacer ◽  
Dna Repeat

We have used the multicopy human rRNA genes as a model system to study replication initiation and termination in mammalian chromosomes. Enrichment for replicating molecules was achieved by isolating S-phase enriched populations of cells by centrifugal elutriation, purification of DNA associated with the nuclear matrix, and a chromatographic procedure that enriches for molecules containing single-stranded regions, a characteristic of replication forks. Two-dimensional agarose gel electrophoresis techniques were used to demonstrate that replication appears to initiate at multiple sites throughout most of the 31-kb nontranscribed spacer (NTS) of human ribosomal DNA but not within the 13-kb transcription unit or adjacent regulatory elements. Although initiation events were detected throughout the majority of the NTS, some regions may initiate more frequently than others. Termination of replication, the convergence of opposing replication forks, was found throughout the ribosomal DNA repeat units, and, in some repeats, specifically at the junction of the 3' end of the transcription unit and the NTS. This site-specific termination of replication is the result of pausing of replication forks near the sites of transcription termination. The naturally occurring multicopy rRNA gene family offers a unique system to study mammalian DNA replication without the use of chemical synchronization agents.

scholarly journals The Role of Metabolites in the Link between DNA Replication and Central Carbon Metabolism in Escherichia coli

Genes ◽  
2020 ◽  
Vol 11 (4) ◽  
pp. 447
Author(s):  
Klaudyna Krause ◽  
Monika Maciąg-Dorszyńska ◽  
Anna Wosinski ◽  
Lidia Gaffke ◽  
Joanna Morcinek-Orłowska ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Dna Replication ◽  
Protein Interactions ◽  
Carbon Metabolism ◽  
Molecular Mechanisms ◽  
Central Carbon Metabolism ◽  
Replication Initiation ◽  
Cell Physiology ◽  
E Coli ◽  
Central Carbon

A direct link between DNA replication regulation and central carbon metabolism (CCM) has been previously demonstrated in Bacillus subtilis and Escherichia coli, as effects of certain mutations in genes coding for replication proteins could be specifically suppressed by particular mutations in genes encoding CCM enzymes. However, specific molecular mechanism(s) of this link remained unknown. In this report, we demonstrate that various CCM metabolites can suppress the effects of mutations in different replication genes of E. coli on bacterial growth, cell morphology, and nucleoid localization. This provides evidence that the CCM-replication link is mediated by metabolites rather than direct protein-protein interactions. On the other hand, action of metabolites on DNA replication appears indirect rather than based on direct influence on the replication machinery, as rate of DNA synthesis could not be corrected by metabolites in short-term experiments. This corroborates the recent discovery that in B. subtilis, there are multiple links connecting CCM to DNA replication initiation and elongation. Therefore, one may suggest that although different in detail, the molecular mechanisms of CCM-dependent regulation of DNA replication are similar in E. coli and B. subtilis, making this regulation an important and common constituent of the control of cell physiology in bacteria.

scholarly journals Coordination between E. coli Cell Size and Cell Cycle Mediated by DnaA

2020 ◽  
Author(s):  
Qing Zhang ◽  
Zhichao Zhang ◽  
Hualin Shi
Keyword(s):  
Cell Cycle ◽  
Growth Rate ◽  
Dna Replication ◽  
Cell Size ◽  
Doubling Time ◽  
Cell Mass ◽  
Replication Initiation ◽  
General Relationship ◽  
E Coli ◽  
Regulatory Processes

Sixty years ago, bacterial cell size was found as an exponential function of growth rate. Fifty years ago, a more general relationship was proposed, in which the cell mass was equal to the initiation mass multiplied by the ratio of the total time of the C and D periods to the doubling time. This relationship has recently been experimentally confirmed by perturbing doubling time, C period, D period or the initiation mass. However, the underlying molecular mechanism remains unclear. Here, we developed a mechanistic and kinetic model to describe how the initiator protein DnaA mediates the initiation of DNA replication in E. coli. In the model, we introduced an initiation probability function involving competitive binding of DnaA-ATP (active) and DnaA-ADP (inactive) at replication origin to determine the initiation of replication. In addition, we considered RNAP availability, ppGpp inhibition, DnaA autorepression, DnaA titration by chromosomal sites, hydrolysis of DnaA-ATP along with DNA replication, reactivation of DnaA-ADP and established a kinetic description of these DnaA regulatory processes. We simulated DnaA kinetics and obtained a self-consistent cell size and a regular DnaA oscillation coordinated with the cell cycle at steady state. The relationship between the cell size obtained by the simulation and the growth rate, C period, D period or initiation mass reproduces the results of the experiment. This model also predicts how the number of DnaA and the initiation mass vary with the perturbation parameters (including those reflecting the mutation or interference of DnaA regulatory processes), which is comparable to experimental data. The results suggest that the regulatory mechanisms of DnaA level and activity are associated with the invariance of initiation mass and the cell size general relationship for matching frequencies of replication initiation and cell division. This study may provide clues for concerted control of cell size and cell cycle in synthetic biology.

scholarly journals Effect of multiple copies ofrpoBCon the rate of RNA synthesis inEscherichia coli

1986 ◽  
Vol 48 (2) ◽  
pp. 61-64 ◽  
Author(s):  
Elena C. Guzman ◽  
Alfonso Jimenez-Sanchez
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Rna Synthesis ◽  
Synthetic Activity ◽  
Gene Products ◽  
Replication Forks ◽  
E Coli ◽  
Multiple Copies ◽  
Autogenous Regulation ◽  
Rate Of Movement

SummaryThe cloning of therpoBandrpoCgenes in a high copy number vector inE. coliincreased the amount of the encoded gene products, the β and β′ subunits of RNA polymerase. However, this unexpectedly caused a 30–50% decrease in RNA synthetic activity which alternatively induced a reduction of growth rate and enlargement of cell size, and decreased the DNA replication time. The results can be explained by autogenous regulation of the RNA polymerase genes by the ββ′ subunits. A relation between the decrease in number of transcription units and the observed higher rate of movement of DNA replication forks is discussed.

scholarly journals Varying Rate of RNA Chain Elongation during rrn Transcription in Escherichia coli

2009 ◽  
Vol 191 (11) ◽  
pp. 3740-3746 ◽  
Author(s):  
P. P. Dennis ◽  
M. Ehrenberg ◽  
D. Fange ◽  
H. Bremer
Keyword(s):  
Rna Polymerase ◽  
Rrna Genes ◽  
Chain Elongation ◽  
23S Rrna ◽  
Physiological Parameter ◽  
E Coli ◽  
16S Gene ◽  
Transcript Elongation ◽  
23S Rrna Genes ◽  
Two Peaks

ABSTRACT The value of the rRNA chain elongation rate in bacteria is an important physiological parameter, as it affects not only the rRNA promoter activity but also the free-RNA polymerase concentration and thereby the transcription of all genes. On average, rRNA chains elongate at a rate of 80 to 90 nucleotides (nt) per s, and the transcription of an entire rrn operon takes about 60 s (at 37°C). Here we have analyzed a reported distribution obtained from electron micrographs of RNA polymerase molecules along rrn operons in E. coli growing at 2.5 doublings per hour (S. Quan, N. Zhang, S. French, and C. L. Squires, J. Bacteriol. 187:1632-1638, 2005). The distribution exhibits two peaks of higher polymerase density centered within the 16S and 23S rRNA genes. An evaluation of this distribution indicates that RNA polymerase transcribes the 5′ leader region at speeds up to or greater than 250 nt/s. Once past the leader, transcription slows down to about 65 nt/s within the 16S gene, speeds up in the spacer region between the 16S and 23S genes, slows again to about 65 nt/s in the 23S region, and finally speeds up to a rate greater than 400 nt/s near the end of the operon. We suggest that the slowing of transcript elongation in the 16S and 23S sections is the result of transcriptional pauses, possibly caused by temporary interactions of the RNA polymerase with secondary structures in the nascent rRNA.

scholarly journals Induction of DNA replication by transcription in the region upstream of the human c-myc gene in a model replication system.

1996 ◽  
Vol 16 (10) ◽  
pp. 5754-5763 ◽  
Author(s):  
R Ohba ◽  
K Matsumoto ◽  
Y Ishimi
Keyword(s):  
Dna Replication ◽  
Rna Polymerase ◽  
Dna Synthesis ◽  
Simian Virus 40 ◽  
Replication Initiation ◽  
T7 Rna Polymerase ◽  
Chain Elongation ◽  
Upstream Region ◽  
Important Relationship ◽  
Myc Gene

An important relationship between transcription and initiation of DNA replication in both eukaryotes and prokaryotes has been suggested. In an attempt to understand the molecular mechanism of this interaction, we examined whether transcription can induce DNA replication in vitro by constructing a system in which both replication and transcription were combined. Relaxed circular DNA possessing a replication initiation zone located upstream of the human c-myc gene and a T7 promoter near the P1 promoter of the gene was replicated in the presence of T7 RNA polymerase. In our model system, replication was carried out with the proteins required for simian virus 40 DNA replication. DNA synthesis, which was dependent on both T7 RNA polymerase and the replication proteins, was detected mainly in the promoter and upstream regions of the c-myc gene. Blocking RNA synthesis at the initial stage of the reaction severely reduced DNA synthesis, suggesting that RNA chain elongation is required to induce DNA synthesis. The results indicated that transcription can induce DNA replication in the upstream region of the transcribed gene, most likely by introducing negative supercoiling into the region, which results in unwinding of the DNA duplex.

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