Regulation Of Chromosome Replication Research Articles

Systemic Deletion of Atp7b Modifies the Hepatocytes’ Response to Copper Overload in the Mouse Models of Wilson Disease

Wilson disease (WD) is a genetic disorder caused by inactivation of the copper (Cu) transporter Atp7b and Cu overload, especially in the liver. Mice with Atp7b deleted either globally (systemic inactivation) or only in hepatocyte recapitulate various aspects of human WD. However, their phenotypes vary, and neither the common response to Cu overload nor factors responsible for phenotypes variability are well defined. Using metabolic, histologic, and proteome analyses in three mouse strains with mutated Atp7b, we show that global inactivation of Atp7b enhances and specifically modifies the hepatocyte response. The loss of Atp7b only in hepatocytes dysregulates lipid and nucleic acid metabolisms and increases the abundance of respiratory chain components and redox balancing enzymes. In global knockouts, independently of the background strain, the metabolism of lipid, nucleic acid, and amino acid is inhibited, respiratory chain components are down-regulated, inflammatory response and regulation of chromosomal replication are enhanced. Decrease in glucokinase and lathosterol oxidase and elevation of mucin-13 and S100A10 are observed in all Atp7b mutant strains and reflects the extent of liver injury. The magnitude of proteome changes in different Atp7b-/- strains and in individual animals inversely correlates with their metallothioneins levels rather than Cu content. These results augment the current model of WD pathogenesis, facilitate the identification of WD-specific markers, and assist the development of new therapies

Systemic deletion of Atp7b modifies the hepatocytes\u2019 response to copper overload in the mouse models of Wilson disease

Wilson disease (WD) is caused by inactivation of the copper transporter Atp7b and copper overload in tissues. Mice with Atp7b deleted either globally (systemic inactivation) or only in hepatocyte recapitulate various aspects of human disease. However, their phenotypes vary, and neither the common response to copper overload nor factors contributing to variability are well defined. Using metabolic, histologic, and proteome analyses in three Atp7b-deficient mouse strains, we show that global inactivation of Atp7b enhances and specifically modifies the hepatocyte response to Cu overload. The loss of Atp7b only in hepatocytes dysregulates lipid and nucleic acid metabolisms and increases the abundance of respiratory chain components and redox balancing enzymes. In global knockouts, independently of their background, the metabolism of lipid, nucleic acid, and amino acids is inhibited, respiratory chain components are down-regulated, inflammatory response and regulation of chromosomal replication are enhanced. Decrease in glucokinase and lathosterol oxidase and elevation of mucin-13 and S100A10 are observed in all Atp7b mutant strains and reflect the extent of liver injury. The magnitude of proteomic changes in Atp7b−/− animals inversely correlates with the metallothioneins levels rather than liver Cu content. These findings facilitate identification of WD-specific metabolic and proteomic changes for diagnostic and treatment.

GammaBOriS: Identification and Taxonomic Classification of Origins of Replication in Gammaproteobacteria using Motif-based Machine Learning

The biology of bacterial cells is, in general, based on information encoded on circular chromosomes. Regulation of chromosome replication is an essential process that mostly takes place at the origin of replication (oriC), a locus unique per chromosome. Identification of high numbers of oriC is a prerequisite for systematic studies that could lead to insights into oriC functioning as well as the identification of novel drug targets for antibiotic development. Current methods for identifying oriC sequences rely on chromosome-wide nucleotide disparities and are therefore limited to fully sequenced genomes, leaving a large number of genomic fragments unstudied. Here, we present gammaBOriS (Gammaproteobacterial oriCSearcher), which identifies oriC sequences on gammaproteobacterial chromosomal fragments. It does so by employing motif-based machine learning methods. Using gammaBOriS, we created BOriS DB, which currently contains 25,827 gammaproteobacterial oriC sequences from 1,217 species, thus making it the largest available database for oriC sequences to date. Furthermore, we present gammaBOriTax, a machine-learning based approach for taxonomic classification of oriC sequences, which was trained on the sequences in BOriS DB. Finally, we extracted the motifs relevant for identification and classification decisions of the models. Our results suggest that machine learning sequence classification approaches can offer great support in functional motif identification.

Reversible lysine acetylation is involved in DNA replication initiation by regulating activities of initiator DnaA in Escherichia coli.

The regulation of chromosomal replication is critical and the activation of DnaA by ATP binding is a key step in replication initiation. However, it remains unclear whether and how the process of ATP-binding to DnaA is regulated. Here, we show that DnaA can be acetylated, and its acetylation level varies with cell growth and correlates with DNA replication initiation frequencies in E. coli. Specifically, the conserved K178 in Walker A motif of DnaA can be acetylated and its acetylation level reaches the summit at the stationary phase, which prevents DnaA from binding to ATP or oriC and leads to inhibition of DNA replication initiation. The deacetylation process of DnaA is catalyzed by deacetylase CobB. The acetylation process of DnaA is mediated by acetyltransferase YfiQ, and nonenzymatically by acetyl-phosphate. These findings suggest that the reversible acetylation of DnaA ensures cells to respond promptly to environmental changes. Since Walker A motif is universally distributed across organisms, acetylation of Walker A motif may present a novel regulatory mechanism conserved from bacteria to eukaryotes.

Aggregation of SeqA protein requires positively charged amino acids in the hinge region

SeqA proteins of Escherichia coli bound to the hemimethylated GATC sequences (hemi-sites) interact with each other and eventually form an aggregate. SeqA foci, which are suggested to be formed by aggregation, play important roles in the regulation of chromosome replication and segregation. We found that aggregation of SeqA proteins was preceded by cooperative interactions between these proteins bound to hemi-sites. Positively charged amino acids in the hinge region, which connects the N-terminal and C-terminal domain of SeqA, were critical for SeqA aggregation on hemimethylated DNA. Although the substitution of positively charged amino acids with negatively charged or neutral amino acids maintained the binding and cooperative interaction of mutant proteins, these proteins were defective in aggregation and foci formation in vitro and in vivo, respectively. Our results suggest that in vivo SeqA foci were formed by aggregation following cooperative interactions between SeqA proteins bound to hemi-sites.

Regulation of chromosomal replication by DnaA protein availability in Escherichia coli: effects of the datA region

Initiation of chromosomal replication in Escherichia coli is dependent on availability of the initiator protein DnaA. We have introduced into E. coli cells plasmids carrying the chromosomal locus datA, which has a high affinity for DnaA. To be able to monitor oriC initiation as a function of datA copy number, we introduced a minichromosome which only replicates from oriC, using a host cell which replicates its chromosome independently of oriC. Our data show that a moderate increase in datA copy number is accompanied by increased DnaA protein synthesis that allows oriC initiation to occur normally, as measured by minichromosome copy number. As datA gene dosage is increased dnaA expression cannot be further derepressed, and the minichromosome copy number is dramatically reduced. Under these conditions the minichromosome was maintained by integration into the chromosome. These findings suggest that the datA locus plays a significant role in regulating oriC initiation, by its capacity to bind DnaA. They also suggest that auto regulation of the dnaA gene is of minor importance in regulation of chromosome initiation.

Regulation of chromosome replication.

The initiation of DNA replication in eukaryotic cells is tightly controlled to ensure that the genome is faithfully duplicated once each cell cycle. Genetic and biochemical studies in several model systems indicate that initiation is mediated by a common set of proteins, present in all eukaryotic species, and that the activities of these proteins are regulated during the cell cycle by specific protein kinases. Here we review the properties of the initiation proteins, their interactions with each other, and with origins of DNA replication. We also describe recent advances in understanding how the regulatory protein kinases control the progress of the initiation reaction. Finally, we describe the checkpoint mechanisms that function to preserve the integrity of the genome when the normal course of genome duplication is perturbed by factors that damage the DNA or inhibit DNA synthesis.

Identification of hemimethylated DNA binding activity in theseqAmutant

A 245 bp segment of E. coli chromosomal replication origin, oriC, contains 11 repeats of the GATC sequence in which adenine is methylated by Dam methylase. Newly replicated oriC is hemimethylated. The parental strand of the newly replicated oriC is methylated, but the nascent strand is not yet methylated until methylated by Dam methylase. The hemimethylated oriC plays an important role in the regulation of chromosomal replication. Activity in the seqA mutant was identified to bind preferentially to hemimethylated DNA, but not to fully‐methylated DNA. This activity may participate in the sequestration of initiation of chromosomal replication

The initiation mess?

This review concerns the mechanisms which control initiation of chromosome replication in enterobacteria with respect to cell growth. Initiation control is commonly separated into positive and negative regulatory mechanisms. Four main points are advanced concerning these different aspects of initiation control. (i) The average concentration of the initiator protein DnaA is proportional to the origin concentration, i.e. the origin per cell mass ratio and, thus, inversely proportional to the very often used term of the 'initiation mass'. (ii) The time of initiation of chromosome replication in the cell cycle is set by DnaA protein accumulating to a threshold level, which in concert with a number of other factors allows for a co-operative formation of the initiation complex. (iii) The time of initiation is not determined by the interaction with these other factors or by the transient interaction between newly replicated origins (oriC) and the cell surface. (iv) The aberrant initiation phenotype observed in various mutants, including dnaA (ts) mutants, might be due to a defective preinitiation DnaA-oriC interaction or it might be due to a defect in the protection of newly initiated origins from reinitiation. Many of these points are discussed and evaluated in view of recent developments concerning the regulation of chromosome replication in Escherichia coli.

Regulation of chromosomal replication in E. coli: Sequestration and beyond

Regulation of chromosomal replication in E. coli: Sequestration and beyond

The regulation of chromosome replication.

The regulation of chromosome replication.

Structure and function of DnaA and the DnaA‐box in eubacteria: evolutionary relationships of bacterial replication origins

DnaA protein (a trans-acting element) and its binding sequence, DnaA-box: (a cis-acting element) are two elements essential for the initiation of chromosomal replication in Escherichia coli and other enteric bacteria. Recently these two elements have been found to be conserved in three Gram-positive bacteria (Bacillus subtilis, Micrococcus luteus and Mycoplasma capricolum) as well as in Gram-negative pseudomonads. DnaA protein was also found to be essential in the initiation of the replication of the B. subtilis chromosome, and regions containing multiple repeats of DnaA-box (DnaA-box region) are found to be active as autonomously replicating elements both in B. subtilis and pseudomonads. In this MicroReview we compare first the structures of these DnaA-box regions and their locations on the chromosome and then functional aspects of DnaA protein and DnaA-box regions in the initiation and regulation of chromosomal replication. From these observations we propose evolutionary relationships between replication origins of eubacteria.

Regulation of chromosomal replication and transcription during early mammalian development.

AbstractMany principles of eukaryotic DNA replication and its relationship to transcription have been revealed by studying the replication of animal virus chromosomes. Now microinjection of viral DNA into eggs and embryos is providing clues about regulation of chromosomal replication and transcription during early mammalian development.

Regulation of Chromosome Replication and the Bacterial Cell Cycle

Regulation of Chromosome Replication and the Bacterial Cell Cycle

Regulation of chromosome replication in Bacillus subtilis: marker frequency analysis after amino acid starvation.

Marker frequency analysis of DNA isolated from amino acid-starved Bacillus subtilis cells shows that most chromosomes have not completed replication to the terminus. This finding agrees with earlier results concerning replication after amino acid starvation in this organism. The results are not compatible with regulation of chromosome replication at the initiation step only, and they suggest that a second regulatory circuit controls replication under conditions of amino acid starvation.

Regulation of chromosome replication in Bacillus subtilis: effects of amino acid starvation in strain W23.

Amino acid starvation allows limited synthesis of deoxyribonucleic acid (DNA) in Bacillus subtilis strain W23. DNA synthesis increased by about 30% after leucine starvation and by about 60% after histidine starvation. Genetic analysis on the DNA synthesized after amino acid starvation showed that all genetic markers examined have replicated, regardless of which amino acid was starved for. Initially, all markers replicated equally, but upon further replication, the thr cysB and the argA to lys regions replicated ahead of their neighboring, proximal regions. This could indicate that preferred stopping sites exist in these regions or additional sites from which replication can originate reside there. The results suggest that chromosome replication continues from those sites where it had stopped during amino acid starvation.

Regulation of chromosome replication in Escherichia coli: a comparison of the effects of phenethyl alcohol treatment with those of amino acid starvation

Escherichia coli 15 T − was treated with phenethyl alcohol at a concentration sufficient eventually to stop DNA synthesis, but not RNA or protein synthesis. DNA synthesis was found to cease at that same region of the chromosome at which it ceases in the absence of required amino acids. It was concluded that phenethyl alcohol allows the completion of a chromosome replication cycle, but prevents the initiation of a new replication cycle. In the presence of phenethyl alcohol, an excess of protein is synthesized which participates in initiating new cycles of DNA replication. Synthesis of this protein is inhibited by chloramphenicol. Although this protein may be necessary for initiating a cycle of DNA replication, it is not sufficient.

Regulation of chromosome replication and segregation in bacteria.

Regulation of chromosome replication and segregation in bacteria.

Regulation of chromosome replication and segregation in bacteria.

Regulation of chromosome replication and segregation in bacteria.

Regulation of chromosome replication in Escherichia coli: Alternate replication of two chromosomes at slow growth rates

Escherichia coli 15 T − , arginine − , methionine − , tryptophan − , grows exponentially with a 40-minute generation period in M9-glucose medium and a 70-minute generation period in M9-succinate medium. The DNA content per cell in these two media is 8·4 and 7·0 × 10 9 daltons, respectively, corresponding to about two chromosomes per cell. Autoradiographic studies demonstrate that, at any instant, cells in glucose cultures are synthesizing about twice as many conserved DNA units as cells in succinate cultures. When starved of amino acids, cells growing in glucose synthesize about 2·5 times as much DNA as cells growing in succinate. Experiments combining a density and a radioactive label demonstrated that in succinate the two chromosomes per cell are synthesized alternately and in sequence. In the absence of amino acids, replication of one chromosome is completed. Replication of the second is initiated upon re-addition of amino acids. Thymine starvation initiates replication prematurely on only those chromosomes which are already in the process of replication. A model for alternate chromosome replication is discussed.