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PubMed1984

Control of replication of bacterial plasmids: genetics, molecular biology, and physiology of the plasmid R1 system.

Plasmids are autonomously replicating DNA molecules that are present in defined copy numbers in bacteria. This number may for some plasmids be very low (2-5 per average cell). In order to be stably inherited, replication and partitioning of the plasmid have to be strictly controlled. Plasmids carry genetic information for both processes. In the present paper we summarize what is known about the replication control system of one low-copy-number plasmid, R1, belonging to the FII incompatibility group. We do so because the FII group seems to be one of the best understood examples with respect to genetics, molecular biology, and physiology of the replication control system. The paper is not a classical review, but rather an essay in which we discuss the aspects of replication control that we regard as being important.

Bacterial Proteins↗
PubMed1992

The level of supercoiling affects the regulation of DNA replication in Escherichia coli.

The chromosome of Escherichia coli is negatively supercoiled. This favours processes that unwind the two DNA strands, such as DNA replication. In this paper, we have investigated the effect of changed levels of overall chromosomal supercoiling on the initiation of DNA replication. Specifically, we have used flow cytometry to reveal effects on the synchrony of initiations of DNA replication in single cells. An increase in the level of supercoiling moderately reduced initiation synchrony. In contrast, decreased supercoiling led to pronounced asynchrony. We have excluded the possibility that this asynchrony is caused by changes in the level of the Dam methyltransferase or the DnaA protein. We suggest that the global level of supercoiling influences the topology of oriC and thereby the sequence of events leading to initiation of DNA replication in E. coli.

Bacterial Proteins↗
PubMed1998

The SbcCD nuclease of Escherichia coli is a structural maintenance of chromosomes (SMC) family protein that cleaves hairpin DNA.

Hairpin structures can inhibit DNA replication and are intermediates in certain recombination reactions. We have shown that the purified SbcCD protein of Escherichia coli cleaves a DNA hairpin. This cleavage does not require the presence of a free (3' or 5') DNA end and generates products with 3'-hydroxyl and 5'-phosphate termini. Electron microscopy of SbcCD has revealed the "head-rod-tail" structure predicted for the SMC (structural maintenance of chromosomes) family of proteins, of which SbcC is a member. This work provides evidence consistent with the proposal that SbcCD cleaves hairpin structures that halt the progress of the replication fork, allowing homologous recombination to restore DNA replication.

Bacterial Proteins↗
PubMed1971

R factor deoxyribonucleic acid in chromosomeless progeny of Escherichia coli.

The deoxyribonucleic acid (DNA) of resistance (R) factor 222 carried by Escherichia coli strain P678-54 was found in the normally chromosomeless progeny (minicells) of that strain. The entry of the R222 DNA into minicells appears to be via segregation at the time of their formation from normal cells. The R222 DNA can replicate in minicells although the extent of its replication appears to be limited. An analysis of the R222 DNA structure indicates that it exists in minicells as double-stranded linear, open circular, and twisted circular monomers (molecular weight, about 6.2 x 10(7) daltons). The monomers visualized by electron microscopy are 31.0 +/- 0.5 mum in length. An examination of the effect of acridine orange on the replication of R222 and colicin E1 DNA indicates the dye intereferes with plasmid DNA replication.

Acridines↗
PubMed2003

Recombination, repair and replication in the pathogenic Neisseriae: the 3 R's of molecular genetics of two human-specific bacterial pathogens.

Most of the detailed mechanisms that have been established for the molecular biological processes that mediate recombination, repair and replication of DNA have come from studies of the Escherichia coli paradigm. The human specific pathogens, Neisseria gonorrhoeae and N. meningitidis, are Gram-negative bacteria that have some molecular processes that are similar to E. coli and others that appear to be divergent. We propose that the pathogenic Neisseriae have evolved a specialized collection of molecular mechanisms to adapt to life limited to human hosts. In this MicroReview, we explore what is known about the basic processes of DNA repair, DNA recombination (genetic exchange and pilin variation) and DNA replication in these human specific pathogens.

Antigens, Bacterial↗
PubMed1984

Initiation of DNA replication in Escherichia coli: RNase H-deficient mutants do not require the dnaA function.

A series of temperature-resistant revertants were isolated from strains of Escherichia coli K12 carrying a temperature-sensitive mutation in the dnaA gene. Four independent revertants were found which still carry the original ts mutation. The ability of these strains to grow at high temperature is due to a suppressor mutation, called sin. All four sin mutations are located between the genes metD and proA on the genetic map of E. coli, which suggests that they all affect the same gene. The sin suppressors, which were isolated for their ability to suppress one dnaA mutation, are also able to suppress three other temperature-sensitive dnaA mutations, but they are not able to suppress mutations in either of the two genes dnaB or dnaC. The sin suppressors alone do not confer any particular phenotype on bacteria, but they are deficient in the enzyme RNase H. On the basis of these findings we propose that the function of the dnaA protein is to protect a DNA-RNA hybrid at the origin of replication against RNase H.

Bacterial Proteins↗
PubMed1970

Concerning the influence of integrated episomes on chromosomal replication in Escherichia coli.

Two strains of Escherichia coli K12, C600, and DG163, in which the origins and directions of DNA replication had been mapped previously, were made lysogenic for lambda and lambdai434. The origins of replication in these lysogenic strains were located with respect to the prophage location by chromosome alignment and subsequent hybridization of (3)H-labeled lambda vir DNA with newly replicated and "old" DNA. The results obtained are consistent with similar origins of replication observed in the nonlysogenic strains. These findings suggest that DNA replication in these cells is under the control of the E. coli chromosomal replicon and is independent of the presence of the lambda replicon as predicted by the Jacob, Brenner, and Cuzin model of DNA replication.

Amino Acids↗
PubMed1991

dnaK protein stimulates a mutant form of dnaA protein in Escherichia coli DNA replication.

Two proteins have been identified which stimulate a mutant form of dnaA protein in replication of plasmids containing the chromosomal origin, oriC. One of these is dnaK protein by the criteria of (i) absence of stimulatory activity in enzyme fractions from dnaK mutants, (ii) elevated levels of stimulatory activity in fractions from a dnaK protein overproducer, (iii) comigration of the stimulatory protein with authentic dnaK protein by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and (iv) replacement of this stimulatory protein by dnaK protein in stimulation assays. The stimulatory effect of dnaK protein on dnaA46 protein in replication suggests that this interaction, occurring prior to its action in DNA replication, may regulate its activity.

Bacterial Proteins↗
PubMed1982

Suppressor mutations (rin) that specifically suppress the recA+ dependence of stable DNA replication in Escherichia coliK-12.

The sdrA102 mutation confers upon cells the ability to replicate DNA in the absence of protein synthesis. This mutation was combined with the recA200 mutation, which renders the recA protein thermolabile, and had little effect on normal replication. However, the sdrA102 recA200 double mutant exhibited temperature-sensitive stable DNA replication: it replicated DNA continuously in the presence of chloramphenicol at 30 degrees C, whereas at 42 degrees C DNA replication ceased after the DNA content increased only 40-45%. Suppressor mutants (rin; recA-independent) capable of stable DNA replication at 42 degrees C were isolated from the double mutant. The suppressor mutant retained all other recA- characteristics, i.e., deficient general recombination, severe UV-sensitivity, and incapability of prophage induction in lysogens. This indicates that the rin mutation specifically suppresses the recA+ dependency of stable DNA replication. It is suggested that the recA+ protein stabilizes a specific structure, similar to an intermediate in recombination, which may function in the initiation of stable DNA replication.

Bacterial Proteins↗
PubMed1988

Transcriptional activation of initiation of replication from the E. coli chromosomal origin: an RNA-DNA hybrid near oriC.

Transcription by RNA polymerase preceding the initiation of replication from the E. coli chromosomal origin (oriC) in vitro enables dnaA protein to open the DNA duplex under conditions when its action alone is insufficient. The RNA polymerases of phages T7 and T3 are as effective as the E. coli enzyme in activating initiation. The persistent RNA transcript hybridized to the template creates an R-loop that is responsible for activation. The activating RNA need not cross oriC, but must be less then 500 bp away. Transcripts lacking a 3' OH group are effective, proving that priming of DNA synthesis is not involved in the activation. Thus, transcription activates the origin of an otherwise inert plasmid by altering the local DNA structure, facilitating its opening by dnaA protein during the assembly of replication forks.

Bacterial Proteins↗
PubMed1995

Replication arrest.

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Bacterial Proteins↗
PubMed1995

Editing DNA replication and recombination by mismatch repair: from bacterial genetics to mechanisms of predisposition to cancer in humans.

A hereditary form of colon cancer, hereditary non-polyposis colon cancer (HNPCC), is characterized by high instability of short repeated sequences known as microsatellites. Because the genes controlling microsatellite stability were known in bacteria and yeast, as was their evolutionary conservation, the search for human genes responsible for HNPCC became a 'targeted' search for known sequences. Mismatch-repair deficiency in bacteria and yeast produces multiple phenotypes as a result of its dual involvement in the editing of both replication errors and recombination intermediates. In addition, mismatch-repair functions are specialized in eukaryotes, characterized by specific mitotic (versus meiotic) functions, and nuclear (versus mitochondrial) localization. Given the number of phenotypes observed so far, we predict other links between mismatch-repair deficiency and human genetic disorders. For example, a similar type of sequence instability has been found in HNPCC tumours and in a number of neuro-muscular genetic disorders. Several human mitochondrial disorders display genomic instabilities reminiscent of yeast mitochondrial mismatch-repair mutants. In general, the process of mismatch repair is responsible for the constant maintenance of genome stability and its faithful transmission from one generation to the next. However, without genetic alteration, species would not be able to adapt to changing environments. It appears that nature has developed both negative and positive controls for genetic diversity. In bacteria, for example, an inducible system (sos) exists which generates genetic alterations in response to environmental stress (e.g. radiation, chemicals, starvation). Hence, the cost of generating diversity to adapt to changing conditions might be paid as sporadic gene alterations associated with disease.

Amino Acid Sequence↗
PubMed2001

Formation of Holliday junctions by regression of nascent DNA in intermediates containing stalled replication forks: RecG stimulates regression even when the DNA is negatively supercoiled.

Replication forks formed at bacterial origins often encounter template roadblocks in the form of DNA adducts and frozen protein-DNA complexes, leading to replication-fork stalling and inactivation. Subsequent correction of the corrupting template lesion and origin-independent assembly of a new replisome therefore are required for survival of the bacterium. A number of models for replication-fork restart under these conditions posit that nascent strand regression at the stalled fork generates a Holliday junction that is a substrate for subsequent processing by recombination and repair enzymes. We show here that early replication intermediates containing replication forks stalled in vitro by the accumulation of excess positive supercoils could be cleaved by the Holliday junction resolvases RusA and RuvC. Cleavage by RusA was inhibited by the presence of RuvA and was stimulated by RecG, confirming the presence of Holliday junctions in the replication intermediate and supporting the previous proposal that RecG could catalyze nascent strand regression at stalled replication forks. Furthermore, RecG promoted Holliday junction formation when replication intermediates in which the replisome had been inactivated were negatively supercoiled, suggesting that under intracellular conditions, the action of RecG, or helicases with similar activities, is necessary for the catalysis of nascent strand regression.

Bacterial Proteins↗
PubMed2006

Structural and physical aspects of bacterial chromosome segregation.

Microscopic observations on the bacterial nucleoid suggest that the chromosome occurs in the cell as a compact nucleoid phase separate from the cytoplasm. Physical theory likewise predicts a phase separation, taking into consideration DNA supercoiling, nucleoid-binding proteins, and excluded-volume interactions between DNA and cytoplasmic proteins. Specific DNA loci, visualized as oriC-GFP spots in the densely packed nucleoid, exhibit a very low diffusion coefficient indicating that they are virtually immobile and may primarily be moved by overall length growth. Such gradual movement could be effectuated by replication, transertion (combined transcription, translation, and insertion of proteins), and actin- (MreB) directed surface synthesis. Differences in the movement and positioning of gene loci between Escherichia coli and Caulobacter crescentus are discussed. We propose that a low diffusion coefficient could explain the linear positioning of genes in the nucleoid and that differential transcriptional activity could induce different mobilities between either replichores (E. coli) or daughter strands (C. crescentus). The transertion process, possibly in combination with MreB cytoskeletal tracks, could overcome the compaction forces and move specific chromosomal regions and the nucleoid as a whole without invoking a dedicated mechanism.

Chromosome Segregation↗
PubMed1995

Effects of the generation of single-stranded DNA on the maintenance of plasmid pMV158 and derivatives in different Bacillus subtilis strains.

The effects of the single-strand origins (SSOs) of plasmid pMV158 on (i) the conversion of its single-stranded (ss) replication intermediates to double-stranded (ds) plasmid DNA and (ii) its maintenance were analyzed. The rolling-circle plasmid pMV158, which replicates via ssDNA intermediates, contains two single-strand origins (SSOs) of replication, palA and palU. In this paper the results obtained with Bacillus subtilis are described; complementary studies with Lactococcus lactis are presented in the accompanying paper (Meijer et al., 1995). While in L. lactis both SSOs are functional as ssDNA conversion signal, only palU appeared to be active B. subtilis. Similar to the situation in L. lactis, the accumulation of large amounts of ssDNA resulted in a severe decrease in plasmid maintenance in B. subtilis. In the latter bacterium large amounts of ssDNA were only accumulated, however, when plasmids lacking a functional SSO were propagated in RecA mutant strains. In wild-type RecA strains these plasmids accumulated only modest amounts of ssDNA and they were maintained at fairly stable levels. The results suggest that in B. subtilis a RecA-mediated alternative pathway exists for the conversion of ssDNA which can improve plasmid maintenance. In addition to ssDNA accumulation and the antagonizing role of RecA therein, two other plasmid regions were shown to affect pMV158 maintenance in B. subtilis. One was the mob gene region, which had a negative effect on plasmid maintenance, and the other the palA type SSO. Although palA was not functional as an ssDNA conversion signal in B. subtilis, its presence had a positive effect on pMV158 maintenance.

Bacillus subtilis↗
PubMed2001

Site-directed mutagenesis and phylogenetic comparisons of the Escherichia coli Tus protein: DNA-protein interactions alone can not account for Tus activity.

The Tus protein of Escherichia coli is capable of arresting DNA replication in an orientation-dependent manner when bound to specific sequences in the bacterial chromosome called Ter sites. Arrest of DNA replication has been postulated to occur either by a barrier mechanism, where Tus acts as a physical block to replication fork progression, or through protein-protein interactions between Tus and some component of the replication fork. A previous mutational analysis of Tus suggested that the amino acids in the L1 loop might play a role in replication arrest. Site-directed mutagenesis of amino acids in the L1 loop and other amino acid residues on the "non-permissive" face of Tus was performed to identify residues that affected Tus function. One mutant, E47Q, gave results that are inconsistent with the barrier model, showing a greater affinity for the Ter site (with a t 1/2 of 348 min versus 150 min for wild-type Tus) but a reduced ability to arrest DNA replication in vivo. In addition to the site-directed mutagenesis studies, the tus genes of Salmonella, Klebsiella, and Yersinia were sequenced and the proteins expressed in E. coli to assess their ability to arrest DNA replication. The results presented here support a role for protein-protein interactions in Tus function, and suggest that residues E47 and E49 participate in replication fork arrest.

Amino Acid Sequence↗
PubMed2006

The Bacillus subtilis primosomal protein DnaD untwists supercoiled DNA.

The essential Bacillus subtilis DnaD and DnaB proteins have been implicated in the initiation of DNA replication. Recently, DNA remodeling activities associated with both proteins were discovered that could provide a link between global or local nucleoid remodeling and initiation of replication. DnaD forms scaffolds and opens up supercoiled plasmids without nicking to form open circular complexes, while DnaB acts as a lateral compaction protein. Here we show that DnaD-mediated opening of supercoiled plasmids is accompanied by significant untwisting of DNA. The net result is the conversion of writhe (Wr) into negative twist (Tw), thus maintaining the linking number (Lk) constant. These changes in supercoiling will reduce the considerable energy required to open up closed circular plectonemic DNA and may be significant in the priming of DNA replication. By comparison, DnaB does not affect significantly the supercoiling of plasmids. Binding of the DnaD C-terminal domain (Cd) to DNA is not sufficient to convert Wr into negative Tw, implying that the formation of scaffolds is essential for duplex untwisting. Overall, our data suggest that the topological effects of the two proteins on supercoiled DNA are different; DnaD opens up, untwists and converts plectonemic DNA to a more paranemic form, whereas DnaB does not affect supercoiling significantly and condenses DNA only via its lateral compaction activity. The significance of these findings in the initiation of DNA replication is discussed.

Bacillus subtilis↗