A novel Escherichia coli mutant capable of DNA replication in the absence of protein synthesis.

An Escherichia coli mutant capable of continued DNA synthesis in the presence of chloramphenicol has been isolated by an autoradiographic technique. The DNA synthesis represents semiconservative replication of E. coli DNA. It can occur in the presence of chloramphenicol or in the absence of essential amino acids, but not in the presence of an RNA synthesis inhibitor, rifampin. The mutant, termed constitutive stable DNA replication (Sdr^c) mutant, appears to grow normally at 37 °C with a slightly slower growth rate than that of the parental strain. DNA replication in the mutant occurs at a reduced rate after 60 minutes in the absence of protein synthesis and continues linearly for several hours thereafter. This distinct slowdown in the DNA replication rate is due to a reduced rate of DNA synthesis in all the cells in the population. Constitutive stable DNA replication appears to require the dnaA and dnaC gene products. The sdr^c mutation has been mapped near the pro-lac region of the E. coli chromosome. The mutation is recessive. Autoradiographic experiments have ruled out the possibility of multiple initiations during a cell cycle. The implication of the above findings is discussed in terms of the regulation of chromosome replication in E. coli.     

Characterization of the replication of Escherichia coli DNA in the absence of protein synthesis: Stable DNA replication

After inhibiting DNA synthesis in Escherichia coli, repeated cycles of chromosome replication can occur in the absence of protein synthesis. This “stable” replication requires the products of all of the known dna genes.Stable replication results from inhibiting DNA synthesis by treatment with naladixic acid, cytosine arabinoside or hydroxyurea; or by placing dnaB, dnaE or dnaG mutants at non-permissive temperatures. It also follows a “shift-up” into rich medium in which RNA and protein are synthesized more rapidly than DNA. Paradoxically, stable replication is induced also by treatment with concentrations of streptolydigin which do not inhibit DNA replication but temporarily and partially inhibit RNA and protein synthesis. During all of these treatments, some protein synthesis must occur.Stable replication is not immediately expressed after a short period of thymine starvation or streptolydigin treatment, but requires a subsequent period of protein synthesis. Once established, however, the stable replication state is permanent and will persist in the absence of protein synthesis or during normal growth.After stable replication has been determined by a period of DNA inhibition, it is possible to inactivate replication by heating dnaA, B, C, E and G temperature-sensitive mutants. However, resynthesis of these gene products in the presence of thymine and at the permissive temperature restores stable replication activity. Since restoration of activity can occur under normal growth conditions which do not induce stable replication, it was concluded that the dnaA, B, C, E and G gene products do not directly determine the stabilized character of the replication fork.A model is presented which attempts to explain the ability of different treatments to induce stable replication.     

Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis

Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) α and δ. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol α–primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.     

Mitochondrial DNA synthesis in cell cycle mutants of saccharomyces cerevisiae

Mitochondrial DNA replication was examined in mutants for seven different Saccharomyces cerevisiae genes which are essential for nuclear DNA replication. In cdc8 and cdc21, mutants defective in continued replication during the S phase of the cell cycle, mitochondrial DNA replication ceases at the nonpermissive temperature. Replication is temperature sensitive even when these mutants are arrested in the G1 phase of the cell cycle with α factor, a condition where mitochondrial DNA replication continues for the equivalent of several generations at the permissive temperature. Therefore the cessation of replication results from a defect in mitochondrial replication per se, rather than from an indirect consequence of cells being blocked in a phase of the cell cycle where mitochondrial DNA is not normally synthesized. Since the temperature-sensitive mutations are recessive, the products of genes cdc8 and cdc21 must be required for both nuclear and mitochondrial DNA replication. In contrast to cdc8 and cdc21, mitochondrial DNA replication continues for a long time at the nonpermissive temperature in five other cell division cycle mutants in which nuclear DNA synthesis ceases within one cell cycle: cdc4, cdc7, and cdc28, which are defective in the initiation of nuclear DNA synthesis, and cdc14 and cdc23, which are defective in nuclear division. The products of these genes, therefore, are apparently not required for the initiation of mitochondrial DNA replication.     

Nucleoside triphosphate dependence of repair replication in toluenized Escherichia coli

Ultraviolet irradiation of Escherichia coli stimulates non-conservative DNA synthesis in cells rendered permeable to nucleoside triphosphates by treatment with toluene. This synthesis, like semi-conservative replication, proceeds in the presence of millimolar concentrations of ATP. Unlike semi-conservative replication, the ultraviolet-stimulated DNA synthesis can proceed if other nucleoside triphosphates are substituted for ATP. The selective dependence of semi-conservative replication upon ATP has been used to study the repair mode of synthesis in the absence of the semi-conservative mode and to demonstrate the dependence of ultraviolet-stimulated synthesis upon the uvrA gene product. Studies with recB mutants show that the nucleoside triphosphate-dependent ultravioletstimulated DNA synthesis occurs in strains deficient in the RecBC deoxyribonuclease.     

In Vivo Occupancy of Mitochondrial Single-Stranded DNA Binding Protein Supports the Strand Displacement Mode of DNA Replication

Mitochondrial DNA (mtDNA) encodes for proteins required for oxidative phosphorylation, and mutations affecting the genome have been linked to a number of diseases as well as the natural ageing process in mammals. Human mtDNA is replicated by a molecular machinery that is distinct from the nuclear replisome, but there is still no consensus on the exact mode of mtDNA replication. We here demonstrate that the mitochondrial single-stranded DNA binding protein (mtSSB) directs origin specific initiation of mtDNA replication. MtSSB covers the parental heavy strand, which is displaced during mtDNA replication. MtSSB blocks primer synthesis on the displaced strand and restricts initiation of light-strand mtDNA synthesis to the specific origin of light-strand DNA synthesis (OriL). The in vivo occupancy profile of mtSSB displays a distinct pattern, with the highest levels of mtSSB close to the mitochondrial control region and with a gradual decline towards OriL. The pattern correlates with the replication products expected for the strand displacement mode of mtDNA synthesis, lending strong in vivo support for this debated model for mitochondrial DNA replication.     

Analysis of Replication Intermediates Indicates That Drosophila melanogaster Mitochondrial DNA Replicates by a Strand-Coupled Theta Mechanism

Mitochondrial DNA synthesis is necessary for the normal function of the organelle and for the eukaryotic organism as a whole. Here we demonstrate, using two-dimensional agarose gel electrophoresis to analyse replication intermediates, that unidirectional, strand-coupled DNA synthesis is the prevalent mode of mtDNA replication in Drosophila melanogaster. Commencing within the single, extended non-coding region (NCR), replication proceeds around the circular genome, manifesting an irregular rate of elongation, and pausing frequently in specific regions. Evidence for a limited contribution of strand-asynchronous DNA synthesis was found in a subset of mtDNA molecules, but confined to the ribosomal RNA gene region, just downstream of the NCR. Our findings imply that strand-coupled replication is widespread amongst metazoans, and should inform future research on mtDNA metabolism in D. melanogaster.     

RecG Directs DNA Synthesis during Double-Strand Break Repair

Homologous recombination provides a mechanism of DNA double-strand break repair (DSBR) that requires an intact, homologous template for DNA synthesis. When DNA synthesis associated with DSBR is convergent, the broken DNA strands are replaced and repair is accurate. However, if divergent DNA synthesis is established, over-replication of flanking DNA may occur with deleterious consequences. The RecG protein of Escherichia coli is a helicase and translocase that can re-model 3-way and 4-way DNA structures such as replication forks and Holliday junctions. However, the primary role of RecG in live cells has remained elusive. Here we show that, in the absence of RecG, attempted DSBR is accompanied by divergent DNA replication at the site of an induced chromosomal DNA double-strand break. Furthermore, DNA double-stand ends are generated in a recG mutant at sites known to block replication forks. These double-strand ends, also trigger DSBR and the divergent DNA replication characteristic of this mutant, which can explain over-replication of the terminus region of the chromosome. The loss of DNA associated with unwinding joint molecules previously observed in the absence of RuvAB and RecG, is suppressed by a helicase deficient PriA mutation (priA300), arguing that the action of RecG ensures that PriA is bound correctly on D-loops to direct DNA replication rather than to unwind joint molecules. This has led us to put forward a revised model of homologous recombination in which the re-modelling of branched intermediates by RecG plays a fundamental role in directing DNA synthesis and thus maintaining genomic stability.     

Efficient long DNA gap-filling in a mammalian cell-free system: A potential new in vitro DNA replication assay

In vitro assay of mammalian DNA replication has been variously approached. Using gapped circular duplex substrates containing a 500-base single-stranded DNA region, we have constructed a mammalian cell-free system in which physiological DNA replication may be reproduced. Reaction of the gapped plasmid substrate with crude extracts of human HeLaS3 cells induces efficient DNA synthesis in vitro. The induced synthesis was strongly inhibited by aphidicolin and completely depended on dNTP added to the system. In cell extracts in which PCNA was depleted step-wise by immunoprecipitation, DNA synthesis was accordingly reduced. These data suggest that replicative DNA polymerases, particularly pol delta, may chiefly function in this system. Furthermore, DNA synthesis is made quantifiable in this system, which enables us to evaluate the efficiency of DNA replication induced. Our system sensitively and quantitatively detected the reduction of the DNA replication efficiency in the DNA substrates damaged by oxidation or UV cross-linking and in the presence of a potent chain terminator, ara-CTP. The quantitative assessment of mammalian DNA replication may provide various advantages not only in basic research but also in drug development.     

Multiple Regulatory Systems Coordinate DNA Replication with Cell Growth in Bacillus subtilis

In many bacteria the rate of DNA replication is linked with cellular physiology to ensure that genome duplication is coordinated with growth. Nutrient-mediated growth rate control of DNA replication initiation has been appreciated for decades, however the mechanism(s) that connects these cell cycle activities has eluded understanding. In order to help address this fundamental question we have investigated regulation of DNA replication in the model organism Bacillus subtilis. Contrary to the prevailing view we find that changes in DnaA protein level are not sufficient to account for nutrient-mediated growth rate control of DNA replication initiation, although this regulation does require both DnaA and the endogenous replication origin. We go on to report connections between DNA replication and several essential cellular activities required for rapid bacterial growth, including respiration, central carbon metabolism, fatty acid synthesis, phospholipid synthesis, and protein synthesis. Unexpectedly, the results indicate that multiple regulatory systems are involved in coordinating DNA replication with cell physiology, with some of the regulatory systems targeting oriC while others act in a oriC-independent manner. We propose that distinct regulatory systems are utilized to control DNA replication in response to diverse physiological and chemical changes.     

Physical studies of RNA involvement in bacteriophage lambda DNA replication and prophage excision

Non-diffusible genetic elements in bacteriophage λ DNA replication and λ prophage excision have been analyzed by the DNA-cutting assay of Freifelder and Kirschner (1971) and Freifelder et al. (1972). The mutant ti12, which affects a unique site for replication in or near the origin of replication (Dove et al., 1971), makes λ DNA partially refractory to replicative DNA-cutting. RNA synthesis in the vicinity of the origin, of replication seems to control the susceptibility of λ DNA to replicative DNA-cutting (Dove et al., 1969). Analogously, RNA synthesis in the vicinity of the left-hand prophage terminus seems to control excisional DNA-cutting of derepressed λ DNA, as predicted by the studies of Davies et al. (1972). These physical studies confirm previous genetic analyses and imply that the elements involved act at a very early stage in replication and in excision.     

Regulation of Deoxyribonucleic Acid Replication and Cell Division in Escherichia coli B/r

Synchronous cultures of Escherichia coli strain B/r were used to investigate the relationship between deoxyribonucleic acid (DNA) replication and cell division. We have determined that terminal steps in division can proceed in the absence of DNA synthesis. Inhibition of DNA replication with nalidixic acid prior to the start of a new round of replication does not stop cell division, which indicates that the start of the round is not essential in triggering cell division. Inhibition of DNA replication at any time prior to the termination of a round of replication completely blocks cell division, which suggests that there may be a link between the end of the replication cycle and the commitment of the cell to divide. Studies that use a temperature-sensitive mutant which is unable to synthesize DNA at the nonpermissive temperature are in complete agreement with those that use nalidixic acid to inhibit DNA synthesis. This adds support to the idea that the treatments employed limit their action to DNA synthesis. Investigation of minicell production indicates that the production of minicells is blocked when DNA synthesis is inhibited with nalidixic acid. Although nuclear segregation is not required for cell division, DNA synthesis is still required to trigger division. The evidence presented suggests strongly that (i) DNA synthesis is essential for cell division, (ii) the end of a round of replication triggers cell division, and (iii) there is considerable time lapse (one-half generation) between the completion of a round of DNA replication and physical separation of the cells.     

Tolerating DNA damage during eukaryotic chromosome replication

In eukaryotes, the evolutionarily conserved RAD6/RAD18 pathway of DNA damage tolerance overcomes unrepaired DNA lesions that interfere with the progression of replication forks, helping to ensure the completion of chromosome replication and the maintenance of genome stability in every cell cycle. This pathway uses two different strategies for damage bypass: translesion DNA synthesis, which is carried out by specialized polymerases that can replicate across the lesions, and DNA damage avoidance, a process that relies on a switch to an undamaged-DNA template for synthesis past the lesion. In this review, we summarise the current knowledge on DNA damage tolerance mechanisms mediated by RAD6/RAD18 that are used by eukaryotic cells to cope with DNA lesions during chromosome replication.     

Recruitment of Orc6l, a dormant maternal mRNA in mouse oocytes, is essential for DNA replication in 1-cell embryos

Mouse oocytes acquire the ability to replicate DNA during meiotic maturation, presumably to ensure that DNA replication does not occur precociously between MI and MII and only after fertilization. Acquisition of DNA replication competence requires protein synthesis, but the identity of the proteins required for DNA replication is poorly described. In Xenopus, the only component missing for DNA replication competence is CDC6, which is synthesized from a dormant maternal mRNA recruited during oocyte maturation, and a similar situation also occurs during mouse oocyte maturation. We report that ORC6L is another component required for acquisition of DNA replication competence that is absent in mouse oocytes. The dormant maternal Orc6l mRNA is recruited during maturation via a CPE present in its 3′ UTR. RNAi-mediated ablation of maternal Orc6l mRNA prevents the maturation-associated increase in ORC6L protein and inhibits DNA replication in 1-cell embryos. These results suggest that mammalian oocytes have more complex mechanisms to establish DNA replication competence when compared to their Xenopus counterparts.     

Low-Molecular-Weight DNA Replication Intermediates in Escherichia coli: Mechanism of Formation and Strand Specificity

Chromosomal DNA replication intermediates, revealed in ligase-deficient conditions in vivo, are of low molecular weight (LMW) independently of the organism, suggesting discontinuous replication of both the leading and the lagging DNA strands. Yet, in vitro experiments with purified enzymes replicating sigma-structured substrates show continuous synthesis of the leading DNA strand in complete absence of ligase, supporting the textbook model of semi-discontinuous DNA replication. The discrepancy between the in vivo and in vitro results is rationalized by proposing that various excision repair events nick continuously synthesized leading strands after synthesis, producing the observed LMW intermediates. Here, we show that, in an Escherichia coli ligase-deficient strain with all known excision repair pathways inactivated, new DNA is still synthesized discontinuously. Furthermore, hybridization to strand-specific targets demonstrates that the LMW replication intermediates come from both the lagging and the leading strands. These results support the model of discontinuous leading strand synthesis in E. coli.     

Inhibition of DNA replication in vitro by pefloxacin

Pefloxacin (a novel quinolone antibiotic) is demonstrated to be a drug inhibiting DNA replication 10-times more efficiently than oxolinic acid measured either in toluene-treated E. coli or in an in vitro replication system for oriC plasmids [6]. DNA repair synthesis is not inhibited by the drug.     

De novo assembly of genuine replication forks on an immobilized circular plasmid in Xenopus egg extracts

We describe an improved model of DNA replication in Xenopus egg extracts, in which a circular plasmid immobilized on paramagnetic beads is used as a template. DNA synthesis occurred on either circular or linear plasmids coupled to the beads, but only DNA synthesis on the circular plasmid was inhibited by geminin and a CDK inhibitor, p21. DNA synthesis on the circular plasmid occurred after a time lag, during which nuclear formation was probably occurring. Although pre-replicative complexes (pre-RCs) were formed soon after mixing plasmids with egg extracts, binding of CDC45, RPA, Pol α, δ and , and PCNA to the circular plasmid was delayed, but still correlated with DNA synthesis. Moreover, p21 inhibited binding of these replication fork proteins to the circular plasmid. Therefore, the circular plasmid, but not the linear plasmid, assembles bona fide replication forks in egg extracts. We conclude that this improved replication system will be useful for studying the mechanism of formation of replication forks in eukaryotic DNA replication.     

Regulation of Cell Division in Escherichia coli

The rate of cell division was measured in cultures of Escherichia coli B/r strain after periods of partial or complete inhibition of deoxyribonucleic acid (DNA) synthesis. The rate of DNA synthesis was temporarily decreased by removing thymidine from the growth medium or replacing it with 5-bromouracil. After restoration of DNA synthesis, a temporary period of accelerated cell division was observed. The results were consistent with the idea that chromosome replication begins when an initiator complement of fixed size accumulated in the cell. The increase in the potential for the initiation of new replication points during inhibition of DNA synthesis results in an increase in the rate of cell division after an interval which encompasses the time for the arrival of these replication points to the termini of the chromosomes and the time from this event to division.