Replication of Epstein–Barr Viral DNA

Epstein–Barr virus (EBV) is a paradigm for human tumor viruses: it is the first virus recognized to cause cancer in people; it causes both lymphomas and carcinomas; yet these tumors arise infrequently given that most people in the world are infected with the virus. EBV is maintained extrachromosomally in infected normal and tumor cells. Eighty-four percent of these viral plasmids replicate each S phase, are licensed, require a single viral protein for their synthesis, and can use two functionally distinct origins of DNA replication, oriP, and Raji ori. Eighty-eight percent of newly synthesized plasmids are segregated faithfully to the daughter cells. Infectious viral particles are not synthesized under these conditions of latent infection. This plasmid replication is consistent with survival of EBV’s host cells. Rare cells in an infected population either spontaneously or following exogenous induction support EBV’s lytic cycle, which is lethal for the cell. In this case, the viral DNA replicates 100-fold or more, uses a third kind of viral origin of DNA replication, oriLyt, and many viral proteins. Here we shall describe the three modes of EBV’s replication as a function of the viral origins used and the viral and cellular proteins that mediate the DNA synthesis from these origins focusing, where practical, on recent advances in our understanding.     

Replication Origins and Timing of Temporal Replication in Budding Yeast: How to Solve the Conundrum?

Similarly to metazoans, the budding yeast Saccharomyces cereviasiae replicates its genome with a defined timing. In this organism, well-defined, site-specific origins, are efficient and fire in almost every round of DNA replication. However, this strategy is neither conserved in the fission yeast Saccharomyces pombe, nor in Xenopus or Drosophila embryos, nor in higher eukaryotes, in which DNA replication initiates asynchronously throughout S phase at random sites. Temporal and spatial controls can contribute to the timing of replication such as Cdk activity, origin localization, epigenetic status or gene expression. However, a debate is going on to answer the question how individual origins are selected to fire in budding yeast. Two opposing theories were proposed: the “replicon paradigm” or “temporal program” vs. the “stochastic firing”. Recent data support the temporal regulation of origin activation, clustering origins into temporal blocks of early and late replication. Contrarily, strong evidences suggest that stochastic processes acting on origins can generate the observed kinetics of replication without requiring a temporal order. In mammalian cells, a spatiotemporal model that accounts for a partially deterministic and partially stochastic order of DNA replication has been proposed. Is this strategy the solution to reconcile the conundrum of having both organized replication timing and stochastic origin firing also for budding yeast? In this review we discuss this possibility in the light of our recent study on the origin activation, suggesting that there might be a stochastic component in the temporal activation of the replication origins, especially under perturbed conditions.     

Biochemical Analysis of DNA Polymerase η Fidelity in the Presence of Replication Protein A

DNA polymerase η (pol η) synthesizes across from damaged DNA templates in order to prevent deleterious consequences like replication fork collapse and double-strand breaks. This process, termed translesion synthesis (TLS), is an overall positive for the cell, as cells deficient in pol η display higher mutation rates. This outcome occurs despite the fact that the in vitro fidelity of bypass by pol η alone is moderate to low, depending on the lesion being copied. One possible means of increasing the fidelity of pol η is interaction with replication accessory proteins present at the replication fork. We have previously utilized a bacteriophage based screening system to measure the fidelity of bypass using purified proteins. Here we report on the fidelity effects of a single stranded binding protein, replication protein A (RPA), when copying the oxidative lesion 7,8-dihydro-8-oxo-guanine(8-oxoG) and the UV-induced cis-syn thymine-thymine cyclobutane pyrimidine dimer (T-T CPD). We observed no change in fidelity dependent on RPA when copying these damaged templates. This result is consistent in multiple position contexts. We previously identified single amino acid substitution mutants of pol η that have specific effects on fidelity when copying both damaged and undamaged templates. In order to confirm our results, we examined the Q38A and Y52E mutants in the same full-length construct. We again observed no difference when RPA was added to the bypass reaction, with the mutant forms of pol η displaying similar fidelity regardless of RPA status. We do, however, observe some slight effects when copying undamaged DNA, similar to those we have described previously. Our results indicate that RPA by itself does not affect pol η dependent lesion bypass fidelity when copying either 8-oxoG or T-T CPD lesions.     

Causation and the Origin of Life. Metabolism or Replication First?

The conceptual gulf that separates the 'metabolism first' and 'replication first' mechanisms for the emergence of life continues to cloud the origin of life debate. In the present paper we analyze this aspect of the origin of life problem and offer arguments in favor of the 'replication first' school. Utilizing Wicken's two-tier approach to causation we argue that a causal connection between replication and metabolism can only be demonstrated if replication would have preceded metabolism. In conjunction with existing empirical evidence and theoretical reasoning, our analysis concludes that there is no substantive evidence for a 'metabolism first' mechanism for life's emergence, while a coherent case can be made for the 'replication first' group of mechanisms. The analysis reaffirms our conviction that life is an extreme expression of kinetic control, and that the emergence of metabolic pathways can be understood by considering life as a manifestation of 'replicative chemistry'.     

Mechanism of Replication of Single-Stranded φX174 DNA VII. Circularization of the Progeny Viral Strand

Linear phiX174 single-stranded DNA can be isolated from phiX phage particles produced under various conditions. About half of the linear strands have a dGMP residue at the 5' end, the remaining have roughly comparable amounts of dCMP, dTMP, and dAMP. The linear strands can be converted to covalently closed circular molecules by polynucleotide ligase, but only after they have been incubated with T4 DNA polymerase and deoxynucleoside triphosphates. Experiments with endonuclease R, the restriction enzyme from Haemophilus influenzae, indicated that the nucleotides incorporated into the DNA during this reaction were found predominantly in a limited region of the genome. The results suggest that the normal intermediate in single-stranded phiX174 DNA synthesis may be a single-stranded linear molecule which is shorter than unit length and is intrinsically capable of circularization.     

Inhibition of Bacteriophage φX174 DNA Replication in dnaB Mutants of Escherichia coli C

Bacteriophage phiX174 DNA replication was examined in temperature-sensitive dnaB mutants of Escherichia coli C to determine which stages require the participation of the product of this host gene. The conversion of the infecting phage single-stranded DNA to the double-stranded replicative form (parental RF synthesis) is completely inhibited at the nonpermissive temperature (41 C) in two of the three dnaB mutants tested. The efficiency of phage eclipse and of phage DNA penetration of these mutant host cells at 41 C is the same as that of the parent host strain. The defect is most likely in the synthesis of the complementary strand DNA. The semiconservative replication of the double-stranded replicative form DNA (RF replication) is inhibited in all three host mutants after shifting from 30 to 41 C. Late in infection, the rate of progeny single-stranded phage DNA synthesis increases following shifts from 30 to 41 C. Approximately the same amounts of phage DNA and of infectious phage particles are made following the shift to 41 C as in the control left at 30 C. The simplest interpretation of our data is that the product of the host dnaB gene is required for phiX174 parental RF synthesis and RF replication, but is not directly involved in phage single-stranded DNA synthesis once it has begun. The possible significance of the synthesis of parental RF DNA at 41 C in one of the three mutants is discussed.     

Self-Propagative Replication of Aβ Oligomers Suggests Potential Transmissibility in Alzheimer Disease

The aggregation of amyloid-β (Aβ) peptide and its deposition in parts of the brain form the central processes in the etiology of Alzheimer disease (AD). The low-molecular weight oligomers of Aβ aggregates (2 to 30 mers) are known to be the primary neurotoxic agents whose mechanisms of cellular toxicity and synaptic dysfunction have received substantial attention in the recent years. However, how these toxic agents proliferate and induce widespread amyloid deposition throughout the brain, and what mechanism is involved in the amplification and propagation of toxic oligomer species, are far from clear. Emerging evidence based on transgenic mice models indicates a transmissible nature of Aβ aggregates and implicates a prion-like mechanism of oligomer propagation, which manifests as the dissemination and proliferation of Aβ toxicity. Despite accumulating evidence in support of a transmissible nature of Aβ aggregates, a clear, molecular-level understanding of this intriguing mechanism is lacking. Recently, we reported the characterization of unique replicating oligomers of Aβ42 (12–24 mers) in vitro called Large Fatty Acid-derived Oligomers (LFAOs) (Kumar et al., 2012, J. Biol. Chem). In the current report, we establish that LFAOs possess physiological activity by activating NF-κB in human neuroblastoma cells, and determine the experimental parameters that control the efficiency of LFAO replication by self-propagation. These findings constitute the first detailed report on monomer – oligomer lateral propagation reactions that may constitute potential mechanism governing transmissibility among Aβ oligomers. These data support the previous reports on transmissible mechanisms observed in transgenic animal models.     

Insights into the Determination of the Templating Nucleotide at the Initiation of φ29 DNA Replication

Bacteriophage φ29 from Bacillus subtilis starts replication of its terminal protein (TP)-DNA by a protein-priming mechanism. To start replication, the DNA polymerase forms a heterodimer with a free TP that recognizes the replication origins, placed at both 5′ ends of the linear chromosome, and initiates replication using as primer the OH-group of Ser-232 of the TP. The initiation of φ29 TP-DNA replication mainly occurs opposite the second nucleotide at the 3′ end of the template. Earlier analyses of the template position that directs the initiation reaction were performed using single-stranded and double-stranded oligonucleotides containing the replication origin sequence without the parental TP. Here, we show that the parental TP has no influence in the determination of the nucleotide used as template in the initiation reaction. Previous studies showed that the priming domain of the primer TP determines the template position used for initiation. The results obtained here using mutant TPs at the priming loop where Ser-232 is located indicate that the aromatic residue Phe-230 is one of the determinants that allows the positioning of the penultimate nucleotide at the polymerization active site to direct insertion of the initiator dAMP during the initiation reaction. The role of Phe-230 in limiting the internalization of the template strand in the polymerization active site is discussed.     

Identification of the Block in the Intracellular Replication of Single-Stranded DNA of Photodynamically Inactivated Bacteriophage φX174

(32)P-labeled single-stranded DNA phage phiX174 was photodynamically inactivated by irradiation in air with visible light in the presence of the acridine dye, proflavine sulfate. The inactivated phages could adsorb to the host cells but failed to lyse them. Formation of intracellular mature phages was almost completely inhibited. Photodynamic lesions in phiX174 DNA caused intracellular formation of defective double-stranded replicative form molecules which ultimately reverted to the single-stranded configuration.     

Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaC Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaC mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. Parental replicative form synthesis is not temperature sensitive. Single-stranded virus DNA continues to be synthesized for at least 45 min after shifting to the nonpermissive temperature late in infection. In contrast, the replication of the replicative form terminates within 5 min after shifting to the nonpermissive temperature.     

Replication of Bacteriophage φX174 DNA in a Temperature-Sensitive dnaE Mutant of Escherichia coli C

Bacteriophage phiX174 cannot grow in a temperature-sensitive dnaE (DNA polymerase III) mutant of Escherichia coli C at the nonpermissive temperature. The inability to grow is the result of inhibition of virus DNA synthesis. The synthesis of the parental replicative form is unaffected, but the replication of the replicative form and the synthesis of the single-stranded virus DNA are inhibited.     

Formation and Stability of the Bacteriophage λ Replication Complexes in UV-Irradiated Escherichia coli

Bacteriophage λ replication complex, containing the phage-encoded O initiator protein protected from proteases by other elements of this complex, is a stable structure that can be inherited by one of the two daughter λ DNA copies after a replication round in Escherichia coli. In normal growth conditions in bacteria bearing a plasmid derived from bacteriophage λ, such a complex may be stable for many cell generations. However, it was found that this stable structure is disassembled under certain conditions, namely, after heat shock. Therefore, we asked whether other environmental stresses may cause disassembly of the λ replication complex. We found that UV irradiation of the host cells prevented formation of the stable λ replication complex (though not preventing phage replication), while the same UV doses did not affect the stability of the replication complex assembled prior to the irradiation. These results indicate that the stable λ replication complex, although sensitive to heat shock, is resistant to some other environmental stresses and that formation of at least two types of λ replication complexes is possible. Both stable and unstable λ replication complexes are functional because replication of λ DNA under conditions preventing formation of the stable complex proceeds efficiently. Received: 18 January 2000 / Accepted: 2 March 2000