DNA replication in Escherichia coli 15T- growing at 20 degrees C.
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Aspartic Acid↗
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Replication of deoxyribonucleic acid in Escherichia coli C mutants temperature sensitive in the initiation of chromosome replication.
An Escherichia coli HF4704S mutant temperature sensitive in deoxyribonucleic acid (DNA) synthesis and different from any previously characterized mutant was isolated. The mutated gene in this strain was designated dnaH. The mutant could grow normally at 27 C but not at 43 C, and DNA synthesis continued for an hour at a decreasing rate and then ceased. After temperature shift-up, the increased amount of DNA was 40 to 50%. When the culture was incubated at 43 C for 70 min and then transferred to 27 C, DNA synthesis resumed after about 50 min, initiating synchronously at a fixed region on the bacterial chromosome. The initiation step in DNA replication sensitive to 30 mug of chloramphenicol per ml occurs synchronously before the resumption of DNA replication after the temperature shift-down, being completed about 30 min before the start of DNA replication. When the cells incubated at 27 C in the presence of 30 mug of chloramphenicol per ml after the temperature shift-down to 27 C were transferred to 43 C with simultaneous removal of the antibiotic, no resumption of DNA replication was observed. When the culture was returned to 43 C after being released from high-temperature inhibition at 30 min before the start of DNA replication, no recovery replication was observed; whereas at 20 min, the recovery of replication was observed. These results indicated that HF4704S was temperature sensitive in the initiation of DNA replication. Analysis of HF4704S, by an interrupted conjugation experiment, indicated that gene dnaH was located at about 64 min on the E. coli C linkage map. In E. coli S1814 (a K-12 derivative), which was a dnaH(ts) transductant from HF4704S (C strain) with phage P1, the mutated gene (dnaH) was demonstrated to be closely linked to the thyA marker by conjugation and P1 transduction experiments and to be distinct from genes dnaA through dnaG.
Chloramphenicol↗
Regulation of IncFII plasmid DNA replication. A quantitative model for control of plasmid NR1 replication in the bacterial cell division cycle.
A quantitative model for the regulation of replication of the low copy number IncFII plasmid NR1 in the Escherichia coli cell division cycle has been developed. The initiation of NR1 replication requires a cis-acting initiator protein whose synthesis is regulated by several mechanisms. The NR1 regulatory processes include co-operative protein-protein interactions in the formation of an active transcription repressor, the interaction of repressor with a rightward operator site in the control of transcription of the initiator gene, and the interaction of an inhibitor RNA transcript with the initiator mRNA in the control of translation of the initiation protein. A statistical thermodynamic model was used to predict probable configurations of the regulatory processes in a single growing cell. These probabilities were coupled by a kinetic model to the events of the cell cycle, such as initiation of mRNA transcription and protein translation, and the initiation of plasmid DNA replication. Parameter values were chosen so that the simulated values for plasmid copy number and the intracellular concentrations of repressor protein and mRNA agreed with experimentally determined estimates. A number of different copy number mutants that have altered one or another of the regulatory processes were simulated by the model. The contributions of each of the regulatory processes toward the overall stability of inheritance of plasmid NR1 in a population of cells in culture were examined. These simulations predict a very stable pattern of inheritance for plasmid NR1 despite its low copy number, in agreement with experimental observation.
Cell Cycle↗
A 27 kd protein of E. coli promotes antitermination of replication in vitro at a sequence-specific replication terminus.
We have discovered a 27 kd protein of E. coli that binds to a terminator site (tau)-terminator protein (ter) complex and abrogates the replication fork-arresting activity of ter protein in vitro. The 27 kd protein also neutralizes the contrahelicase activity of ter protein, allowing dnaB helicase to unwind DNA past a tau-ter complex. The stimulatory activity of low levels of ter protein on helicase II is also abolished by the 27 kd protein. The binding of the 27 kd protein to a tau-ter complex does not appear to dissociate the ter protein from the DNA. Although the in vivo function of the 27 kd protein is unknown at this time, it has the major attributes of a novel replication antiterminator in vitro.
Bacterial Proteins↗
Nature of the SOS-inducing signal in Escherichia coli. The involvement of DNA replication.
The SOS genes of Escherichia coli, which include many DNA repair genes, are induced by DNA damage. Although the central biochemical event in induction, activation of RecA protein through binding of single-stranded DNA and ATP to promote cleavage of the LexA repressor, is known, the cellular event that provides this activation following DNA damage has not been well understood. We provide evidence here that the major pathway of induction after damage by a typical agent, ultraviolet light, requires an active replication fork; this result supports the model that DNA replication leaves gaps where elongation stops at damage-induced lesions, and thus provides the single-stranded DNA that activates RecA protein. In order to detect quantitatively the immediate product of the inducing signal, activated RecA protein, we have designed an assay to measure the rate of disappearance of intact LexA repressor. With this assay, we have studied the early phase of the induction process. LexA cleavage is detectable within minutes after DNA damage and occurs in the absence of protein synthesis. By following the reaccumulation of LexA in the cell, we detect repair of DNA and the disappearance of the inducing signal. Using this assay, we have measured the LexA content of wild-type and various mutant cells, characterized the kinetics and conditions for development of the inducing signal after various inducing treatments and, finally, have shown the requirement for DNA replication in SOS induction by ultraviolet light.
Bacterial Proteins↗
Selection for chromosome architecture in bacteria.
Bacterial chromosomes are immense polymers whose faithful replication and segregation are crucial to cell survival. The ability of proteins such as FtsK to move unidirectionally toward the replication terminus, and direct DNA translocation into the appropriate daughter cell during cell division, requires that bacterial genomes maintain an architecture for the orderly replication and segregation of chromosomes. We suggest that proteins that locate the replication terminus exploit strand-biased sequences that are overrepresented on one DNA strand, and that selection increases with decreased distance to the replication terminus. We report a generalized method for detecting these architecture imparting sequences (AIMS) and have identified AIMS in nearly all bacterial genomes. Their increased abundance on leading strands and decreased abundance on lagging strands toward replication termini are not the result of changes in mutational bias; rather, they reflect a gradient of long-term positive selection for AIMS. The maintenance of the pattern of AIMS across the genomes of related bacteria independent of their positions within individual genes suggests a well-conserved role in genome biology. The stable gradient of AIMS abundance from replication origin to terminus suggests that the replicore acts as a target of selection, where selection for chromosome architecture results in the maintenance of gene order and in the lack of high-frequency DNA inversion within replicores.
Base Sequence↗
[The duplification system of the bacterial cell. I. Relation between DNA replication and cell division under the conditions of balanced growth].
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Bacillus subtilis↗
Interplay between DnaA and SeqA proteins during regulation of bacteriophage lambda pR promoter activity.
DnaA and SeqA proteins are main regulators (positive and negative, respectively) of the chromosome replication in Escherichia coli. Nevertheless, both these replication regulators were found recently to be also transcription factors. Interestingly, both DnaA and SeqA control activity of the bacteriophage lambdap(R) promoter by binding downstream of the transcription start site, which is unusual among prokaryotic systems. Here we asked what are functional relationships between these two transcription regulators at one promoter region. Both in vivo and in vitro studies revealed that DnaA and SeqA can activate the p(R) promoter independently and separately rather than in co-operation, however, increased concentrations of one of these proteins negatively influenced the transcription stimulation mediated by the second regulator. This may suggest a competition between DnaA and SeqA for binding to the p(R) regulatory region. The physiological significance of this DnaA and SeqA-mediated regulation of p(R) is demonstrated by studies on lambda plasmid DNA replication in vivo.
Bacterial Outer Membrane Proteins↗
[Various aspects of the problem of chromosomal DNA replication].
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Animals↗
Differential effects of myeloperoxidase-derived oxidants on Escherichia coli DNA replication.
The microbicidal myeloperoxidase (MPO)-H2O2-chloride system strongly inhibits Escherichia coli DNA synthesis. Also, cell envelopes from MPO-treated E. coli cells lose their ability to interact with hemimethylated DNA sequences of oriC, the chromosomal origin of replication, raising the prospect that suppression of DNA synthesis involves impairment of oriC-related functions (H. Rosen, et al. Proc. Natl. Acad. Sci. USA, 87:10048-10052, 1990). To evaluate whether origin-specific DNA sequences play a role in the MPO effect on E. coli DNA synthesis, plasmid DNA replication was compared to total (chromosomal) DNA replication for six plasmids with three distinct origins of replication. Plasmid pCM700 replication, replicating from oriC, was as sensitive to MPO-mediated inhibition as was total (chromosomal) DNA replication. A regression line describing this relationship had a slope of 0.90, and the r2 was 0.89. In contrast, the replication activities of three of four non-oriC plasmids, pUC19, pACYC184, and pSC101, demonstrated significant early resistance to inhibition by MPO-derived oxidants. The exception to this resistance pattern was plasmid pSP102, which has an origin derived from P1 phage. pSP102 replication declined similarly to that of total DNA synthesis. The regression line for pSP102 replication versus total DNA synthesis had a slope of 0.95, and the r2 was 0.92. The biochemical requirements for P1-mediated replication are strikingly similar to those for oriC-mediated replication. It is proposed that one of these requirements, common to oriC and the P1 origin but not critical to the replication of the other non-oriC plasmids, is an important target for MPO-mediated oxidations that mediate the initial decline in E. coli chromosomal DNA synthesis.
Chromosomes, Bacterial↗
Functionality of the dnaA protein binding site in DNA replication is orientation-dependent.
We analyzed the functionality of different dnaA protein binding sites by assaying in vitro dnaA-dependent replication of pBR322 derivatives. Single dnaA sites from oriC and from the mioC and dnaA gene promoters were active when combined with the primer generating element of pBR322 in a proper distance. Prereplisome assembly did not require sequences in addition to the 9-base pair consensus dnaA binding site. Inversion of the structurally asymmetric dnaA site relative to its orientation in wild type pBR322 resulted in a marked reduction in replication efficiency, as observed with five different dnaA sites studied. The direction of DNA replication was not affected.
Bacterial Proteins↗
[The Escherichia coli cell cycle].
This review summarizes present knowledge of the bacterial cell cycle with particular emphasis on Escherichia coli. We discuss data coming from three different types of approaches to the study of cell extension and division: The search for discrete events occurring once per division cycle. It is generally agreed that the initiation and termination of DNA replication and cell septation are discrete events; there is less agreement on the sudden doubling in rate of cell surface extension, murein biosynthesis and the synthesis of membrane proteins and phospholipids. We discuss what is known about the temporal relationship amongst the various cyclic events studied. The search for discrete growth zones in the cell envelope layers. We discuss conflicting reports on the existence of murein growth zones and protein insertion sites in the inner and outer membranes. Elucidation of the mechanism regulating the initiation of DNA replication. The concept of "critical initiation mass" is examined. We review data suggesting that the DNA is attached to the envelope and discuss the role of the latter in the initiation of DNA replication.
Bacterial Proteins↗
Rolling-circle replication of bacterial plasmids.
Many bacterial plasmids replicate by a rolling-circle (RC) mechanism. Their replication properties have many similarities to as well as significant differences from those of single-stranded DNA (ssDNA) coliphages, which also replicate by an RC mechanism. Studies on a large number of RC plasmids have revealed that they fall into several families based on homology in their initiator proteins and leading-strand origins. The leading-strand origins contain distinct sequences that are required for binding and nicking by the Rep proteins. Leading-strand origins also contain domains that are required for the initiation and termination of replication. RC plasmids generate ssDNA intermediates during replication, since their lagging-strand synthesis does not usually initiate until the leading strand has been almost fully synthesized. The leading- and lagging-strand origins are distinct, and the displaced leading-strand DNA is converted to the double-stranded form by using solely the host proteins. The Rep proteins encoded by RC plasmids contain specific domains that are involved in their origin binding and nicking activities. The replication and copy number of RC plasmids, in general, are regulated at the level of synthesis of their Rep proteins, which are usually rate limiting for replication. Some RC Rep proteins are known to be inactivated after supporting one round of replication. A number of in vitro replication systems have been developed for RC plasmids and have provided insight into the mechanism of plasmid RC replication.
Amino Acid Sequence↗
The inactivated plasmid inititator protein RepC/RepC* may have a regulatory role.
During replication of the plasmid pT181, the initiator protein RepC is modified by the addition of an oligodeoxynucleotide, giving rise to a new form, RepC*. Here we show that during in vitro replication, RepC* is radioactively labeled, suggesting that the source of the RepC* oligodeoxynucleotide is the newly synthesized pT181 DNA. The RepC/RepC* heterodimer retains its ability to bind the pT181 double-strand origin and, therefore, it may act as a competitive inhibitor of the RepC homodimer during replication.
Bacterial Proteins↗
The great GATC: DNA methylation in E. coli.
In Escherichia coli the methylation of the adenine in the sequence 5'-GATC-3' is catalysed by the dam gene product, a DNA adenine methylase. We review the proposed roles for this methylation, and the sequence it modifies, in mismatch repair, DNA-protein interaction, gene expression, the initiation of chromosome replication, chromosome segregation, chromosome structure and the occurrence of mutational hotspots.
Base Sequence↗
Plasmid pT181 replication is decreased at high levels of RepC per plasmid copy.
The replication of staphylococcal plasmid pT181 is indirectly controlled at the level of the synthesis of its replication initiator, RepC. As a result, high levels of RepC synthesis per plasmid copy were expected to lead to autocatalytic plasmid replication, which secondarily would affect host physiology. Surprisingly, RepC overexpression was found to lead to a rapid decrease in pT181 copy number and replication rate. These effects depended on the ratio of RepC to the pT181 replication origin rather than on the absolute amount of RepC in the cell. In a wild-type host, the increase in RepC/plasmid copy also inhibited chromosome replication and cell division. The changes in host physiology did not play any role in the decrease in pT181 replication caused by RepC overexpression since pT181 replication responded in the same way in a host mutant insensitive to the effects of RepC induction. These results suggest that pT181, the prototype of an entire class of plasmids from Gram-positive bacteria, responds to overexpression of its replication initiator by a decrease in plasmid replication.
Bacterial Proteins↗
Recombinational rescue of the stalled DNA replication fork: a model based on analysis of an Escherichia coli strain with a chromosome region difficult to replicate.
To examine the physiological effects of DNA replication arrest at the terminus (Ter), we constructed a replication-blocked Escherichia coli strain so that both bidirectional replication forks would be impeded at two flanking Ter sites, one artificial and the other natural. While the blocked strain grew slightly more slowly than a control strain, it had abnormal phenotypes similar to those of E. coli dam mutants, i.e., hyper-Rec phenotype, recA(+)- and recB+ (C+)-dependent growth, and constitutive SOS induction. The observation that these two apparently unrelated mutants cause similar phenotypes led us to design a model. We propose that the following sequential events may occur in both strains. A double-strand (ds) break occurs at the blocked replication fork in the blocked strain and at the ongoing fork in the dam mutant, through which RecBCD enzyme enters and degrades the ds DNA molecule, and the degradation product serves as the signal molecule for SOS induction. When RecBCD enzyme meets an appropriately oriented Chi sequence, its DNase activity is converted to recombinase enzyme, which is able to repair the ds end, recombinationally. this model (i) explains the puzzling phenotype of recA and recB (C) mutants and the SOS-inducing phenotype of polA, lig, and dna mutants under restrictive conditions, (ii) provides an interpretation for the role of the Chi sequence, and (iii) suggests a possible key role for homologous recombination with regard to cell survival following the arrest of DNA replication.
Chromosomes, Bacterial↗
Anti-hepatitis B virus activities of purine derivatives of oxetanocin A.
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Adenine↗