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.     

3′ cis-Acting Elements That Contribute to the Competence and Efficiency of Japanese Encephalitis Virus Genome Replication: Functional Importance of Sequence Duplications,Deletions, and Substitutions

The positive-strand RNA genome of Japanese encephalitis virus (JEV) terminates in a highly conserved 3′-noncoding region (3′NCR) of six domains (V, X, I, II-1, II-2, and III in the 5′-to-3′ direction). By manipulating the JEV genomic RNA, we have identified important roles for RNA elements present within the 574-nucleotide 3′NCR in viral replication. The two 3′-proximal domains (II-2 and III) were sufficient for RNA replication and virus production, whereas the remaining four (V, X, I, and II-1) were dispensable for RNA replication competence but required for maximal replication efficiency. Surprisingly, a lethal mutant lacking all of the 3′NCR except domain III regained viability through pseudoreversion by duplicating an 83-nucleotide sequence from the 3′-terminal region of the viral open reading frame. Also, two viable mutants displayed severe genetic instability; these two mutants rapidly developed 12 point mutations in domain II-2 in the mutant lacking domains V, X, I, and II-1 and showed the duplication of seven upstream sequences of various sizes at the junction between domains II-1 and II-2 in the mutant lacking domains V, X, and I. In all cases, the introduction of these spontaneous mutations led to an increase in RNA production that paralleled the level of protein accumulation and virus yield. Interestingly, the mutant lacking domains V, X, I, and II-1 was able to replicate in hamster BHK-21 and human neuroblastoma SH-SY5Y cells but not in mosquito C6/36 cells, indicating a cell type-specific restriction of its viral replication. Thus, our findings provide the basis for a detailed map of the 3′ cis-acting elements in JEV genomic RNA, which play an essential role in viral replication. They also provide experimental evidence for the function of 3′ direct repeat sequences and suggest possible mechanisms for the emergence of these sequences in the 3′NCR of JEV and perhaps in other flaviviruses.Japanese encephalitis virus (JEV), a mosquito-borne flavivirus of the family Flaviviridae, is serologically related to several significant human pathogens, including West Nile virus (WNV), Kunjin virus (KUNV), St. Louis encephalitis virus, and Murray Valley encephalitis virus. It is also phylogenetically close to other clinically important human pathogens, including yellow fever virus (YFV) and dengue virus (DENV) (11, 67). JEV is the leading cause of viral encephalitis in Southeast Asia, including China, Japan, Korea, the Philippines, Thailand, and India, and it has begun to expand throughout the Indonesian archipelago and as far as Australia (21, 43). Despite the fact that JEV is generally asymptomatic, ∼50,000 cases are reported annually, and the disease has a mortality rate of ∼25%, mainly in children and young adults (29, 63). Thus, the geographic expansion and clinical importance of JEV infection have drawn increasing attention from the international public health community (44, 71).Like other flaviviruses, JEV is a spherical enveloped virus (∼50 nm diameter) with a single-stranded positive-sense RNA genome that contains a 5′ cap structure but lacks a 3′ polyadenylated tail. Its genomic RNA of ∼11,000 nucleotides (nt) consists of a single long open reading frame (ORF) with two noncoding regions (NCRs) at the 5′ and 3′ ends (41, 84). The ORF is translated into an ∼3,400-amino acid polyprotein precursor, which is co- or posttranslationally cleaved by a cellular protease(s) or a viral protease complex into 10 mature proteins: (i) three structural proteins, the capsid (C), premembrane (prM; which is further processed into pr and M), and envelope (E) proteins; and (ii) seven nonstructural proteins, NS1, NS2A, NS2B, NS3, NS4A, NS4B, and NS5, as arranged in the genome (13, 41, 84). The nonstructural proteins, together with cellular factors, form a viral replicase complex that directs the replication of the genomic RNA in the cytoplasm of the host cell in association with perinuclear membranes (40, 74). For the synthesis of the genomic RNA to take place, this replicase complex must specifically recognize viral cis-acting RNA elements, defined by primary sequences or secondary/tertiary structures. These RNA elements are found in various locations within the genome but most frequently are located in the 5′- and 3′NCRs (23, 47). The identification and characterization of these cis-acting RNA elements is critical for understanding the complete cycle of JEV genome replication.The availability of the complete nucleotide sequence of YFV genomic RNA (57) has led to the identification of three major conserved elements in the 5′- and 3′-terminal regions of the genomic RNA that contain the short primary sequences and secondary structures required for flavivirus RNA replication. (i) Both ends of the genomic RNA terminate with the conserved dinucleotides 5′-AG and CU-3′ (9, 10, 32, 45, 57, 72, 73) in all flaviviruses except an insect cell fusing agent virus (12). Mutations substituting another nucleotide for one of these four nucleotides in KUNV or WNV replicon RNA are known to abolish or compromise RNA replication (35, 69). (ii) A 3′ stem-loop structure (3′SL) has been recognized in all flaviviruses within the ∼90-nt 3′-terminal region of the genomic RNA (9, 45, 57). The structural and functional importance of this 3′SL in RNA replication has been demonstrated in several flaviviruses (9, 18, 49, 50, 61, 70, 82, 86). (iii) The presence of short 5′ and 3′ cyclization sequences (5′CYC and 3′CYC, respectively) in all mosquito-borne flaviviruses suggests that flavivirus genomes can cyclize via 5′-3′ long-range base-pairing interaction, since the 3′CYC upstream of the 3′SL is complementary to the 5′CYC in the 5′ coding region of the C protein (30). The role of these CYC motifs in RNA replication has been well characterized via cell-based assays in many mosquito-borne flaviviruses, including KUNV (34), WNV (42), YFV (8, 14), and DENV (2, 22, 49), and in cell-free systems in the case of WNV (51) and DENV (1, 3, 79, 80). Other RNA elements that have recently been shown to be important for RNA replication in DENV and WNV include an additional pair of complementary sequences (designated 5′- and 3′UARs) that participate in genome cyclization (3, 4, 17, 87) and a 5′ stem-loop structure (designated 5′SLA) present within the 5′NCR that promotes RNA synthesis in association with the 3′NCR (22).In all flaviviruses, the 3′NCR of the genomic RNA is relatively long (∼400 to ∼800 nt), with an array of conserved primary sequences and secondary structures. Although significant progress has been made in identifying cis-acting elements within the 3′NCRs that are essential for RNA replication, most of these elements (i.e., the 3′CYC, 3′SL, and CU-3′) are limited to the ∼100-nt 3′-terminal region that is highly conserved in these viruses (see recent reviews in references 23 and 47). However, the functional importance of the remaining 5′-proximal region of the 3′NCR, which differs in sequence between the various serological groups, is poorly understood. In particular, comparative sequence analyses and genetic algorithm-based computer modeling have suggested that in addition to the well-studied ∼100-nt 3′-proximal region, the remaining ∼474-nt 5′-proximal region of the 574-nt JEV 3′NCR also contains several RNA elements that may play critical roles in the viral life cycle (52, 55, 56, 68). To date, however, experimental evidence for the functional importance of these potential RNA elements in JEV genomic RNA replication is lacking.In the present study, we have identified and characterized the 3′ cis-acting RNA elements within the JEV 3′NCR and shown that they play an essential and/or regulatory role in genomic RNA replication. In particular, we have constructed and functionally characterized genome-length JEV mutant cDNAs with a series of 5′-to-3′ or 3′-to-5′ progressive deletions within the 3′NCR. In addition to identifying particular mutations within this region that affect either the competence or efficiency of genomic RNA replication, we found that the serial passaging of these mutants in susceptible BHK-21 cells produced a large number of pseudorevertants bearing a wide variety of spontaneous point mutations and sequence duplications, some of which were capable of restoring the replication competence of the defective mutants or enhancing replication efficiency. In addition, we assessed the replication of these mutants in three different cell types (BHK-21, SH-SY5Y, and C6/36 cells). Collectively, these data offer new insights into the functional importance of 3′ cis-acting RNA elements that regulate the cell type-dependent replication of JEV and perhaps other closely related mosquito-borne flaviviruses. Our findings also provide experimental evidence for the emergence of functional 3′ direct repeat sequences that are duplicated from the coding region and 3′NCR of JEV genomic RNA.     

The Coordination of Centromere Replication,Spindle Formation,and Kinetochore–Microtubule Interaction in Budding Yeast

The kinetochore is a protein complex that assembles on centromeric DNA to mediate chromosome–microtubule interaction. Most eukaryotic cells form the spindle and establish kinetochore–microtubule interaction during mitosis, but budding yeast cells finish these processes in S-phase. It has long been noticed that the S-phase spindle in budding yeast is shorter than that in metaphase, but the biological significance of this short S-phase spindle structure remains unclear. We addressed this issue by using ask1-3, a temperature-sensitive kinetochore mutant that exhibits partially elongated spindles at permissive temperature in the presence of hydroxyurea (HU), a DNA synthesis inhibitor. After exposure to and removal of HU, ask1-3 cells show a delayed anaphase entry. This delay depends on the spindle checkpoint, which monitors kinetochore–microtubule interaction defects. Overproduction of microtubule-associated protein Ase1 or Cin8 also induces spindle elongation in HU-arrested cells. The spindle checkpoint-dependent anaphase entry delay is also observed after ASE1 or CIN8 overexpression in HU-arrested cells. Therefore, the shorter spindle in S-phase cells is likely to facilitate proper chromosome–microtubule interaction.     

Heat Induction of Prophage φ105 in Bacillus subtilis: Replication of the Bacterial and Bacteriophage Genomes

A temperature-inducible mutant of temperate Bacillus bacteriophage phi105 was isolated and used to lysogenize a thymine-requiring strain of Bacillus subtilis 168. Synthesis of phage and bacterial deoxyribonucleic acid (DNA) was studied by sucrose gradient centrifugation and density equilibrium centrifugation of DNA extracted from induced bacteria. The distribution of DNA in the gradients was measured by differential isotope and density labeling of DNA before and after induction and by measuring the biological activity of the DNA in genetic transformation, in rescue of phage markers, and in infectivity assays. At early times after induction, but after at least one round of replication, phage DNA remains associated with high-molecular-weight DNA, whereas, later in the infection, phage DNA is associated with material of decreasing molecular weight. Genetic linkage between phage and bacterial markers can be demonstrated in replicated DNA from induced cells. Prophage induction is shown to affect replication of the bacterial chromosome. The overall rate of replication of prelabeled bacterial DNA is identical in temperature-induced lysogenics and in "mock-induced" wild-type phi105 lysogenics. The rate of replication of the bacterial marker phe-1 (and also of nia-38), located close to the prophage in direction of the terminus of the bacterial chromosome, is increased in induced cells, however, relative to other bacterial markers tested. In temperature-inducible lysogenics, where the prophage also carries a ts mutation which blocks phage DNA synthesis, replication of both phage and bacterial DNA stops after about 50% of the phage DNA has replicated once. The results of these experiments suggest that the prophage is not initially excised in induced cells, but rather it is specifically replicated in situ together with adjacent parts of the bacterial chromosome.     

National politics and international trends: EMBO and the making of molecular biology in Spain (1960–1975)

From the mid-1960s onwards, a set of Spanish molecular biology research groups emerged in Spain. The factors contributing to this included: the return of a group of molecular biologists from their postdoctoral period abroad, the negotiations for the return of Spanish-born Nobel prize winner Severo Ochoa from New York, the negotiations for Spanish membership in the European Conference of Molecular Biology, and national policy towards university reform. As a result, the early molecular biologists’ research groups began to be recognised as research schools by Spanish authorities and postgraduate courses and new research centres for molecular biology were set up. Foreign influence in the whole process was crucial.     

DNA Methylation and Replication: Implications for the “Deletion Hotspot” Region of FMR1

Expansion and hyper-methylation of a CGG repeat tract are the main causes of fragile X syndrome (FRAXA). In some rare instances, FRAXA patients harbor not only an expanded CGG tract, but a deletion encompassing the CGG repeat and flanking sequences as well. Through the use of an SV40 primate replication system, it was possible to determine that CpG methylation and DNA replication may actually mediate the formation of these rare events. Also, the genetically stabilizing AGG interruptions can be lost by replication-mediated CGG deletions.     

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'.     

Blood Amyloid Beta Levels in Healthy,Mild Cognitive Impairment and Alzheimer’s Disease Individuals: Replication of Diastolic Blood Pressure Correlations and Analysis of Critical Covariates

Plasma amyloid beta (Aβ) levels are being investigated as potential biomarkers for Alzheimer’s disease. In AB128 cross-sectional study, a number of medical relevant correlates of blood Aβ40 or Aβ42 were analyzed in 140 subjects (51 Alzheimer’s disease patients, 53 healthy controls and 36 individuals diagnosed with mild cognitive impairment). We determined the association between multiple variables with Aβ40 and Aβ42 levels measured in three different blood compartments called i) Aβ directly accessible (DA) in the plasma, ii) Aβ recovered from the plasma matrix (RP) after diluting the plasma sample in a formulated buffer, and iii) associated with the remaining cellular pellet (CP). We confirmed that diastolic blood pressure (DBP) is consistently correlated with blood DA Aβ40 levels (r=-0.19, P=0.032). These results were consistent in the three phenotypic groups studied. Importantly, the observation resisted covariation with age, gender or creatinine levels. Observed effect size and direction of Aβ40 levels/DBP correlation are in accordance with previous reports. Of note, DA Aβ40 and the RP Aβ40 were also strongly associated with creatinine levels (r=0.599, P<<0.001) and to a lesser extent to urea, age, hematocrit, uric acid and homocysteine (p<0.001). DBP and the rest of statistical significant correlates identified should be considered as potential confounder factors in studies investigating blood Aβ levels as potential AD biomarker. Remarkably, the factors affecting Aβ levels in plasma (DA, RP) and blood cell compartments (CP) seem completely different.     

Role of STN1 and DNA Polymerase α in Telomere Stability and Genome-Wide Replication in Arabidopsis

The CST (Cdc13/CTC1-STN1-TEN1) complex was proposed to have evolved kingdom specific roles in telomere capping and replication. To shed light on its evolutionary conserved function, we examined the effect of STN1 dysfunction on telomere structure in plants. STN1 inactivation in Arabidopsis leads to a progressive loss of telomeric DNA and the onset of telomeric defects depends on the initial telomere size. While EXO1 aggravates defects associated with STN1 dysfunction, it does not contribute to the formation of long G-overhangs. Instead, these G-overhangs arise, at least partially, from telomerase-mediated telomere extension indicating a deficiency in C-strand fill-in synthesis. Analysis of hypomorphic DNA polymerase α mutants revealed that the impaired function of a general replication factor mimics the telomeric defects associated with CST dysfunction. Furthermore, we show that STN1-deficiency hinders re-replication of heterochromatic regions to a similar extent as polymerase α mutations. This comparative analysis of stn1 and pol α mutants suggests that STN1 plays a genome-wide role in DNA replication and that chromosome-end deprotection in stn1 mutants may represent a manifestation of aberrant replication through telomeres.     

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     

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 mutants of the F-plasmid of Escherichia coli K-12: I. Isolation and characterization of temperature-sensitive replication mutants of F′-gal+

Eighteen mutants that are temperature-sensitive for vegetative replication (rep) were isolated from two F′-gal⁺ plasmids (F8 and F8-4) after N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis. Some of the mutants also have reduced transfer ability at both permissive and nonpermissive temperatures. Plasmid-plasmid P1 transduction has revealed that in some instances, the altered transfer ability is located in the transfer operon and is distinct from the rep mutation. However, in other cases, the replication and transfer defects have not been separated by P1 transduction. The implications of these results for the relationship between vegetative DNA replication and DNA replication during conjugation are discussed. In vivo recombinational results suggested that the temperature-sensitive mutations were not located in the same regions of the two F′-plasmids. We confirmed that no inversion, secondary deletion, or translocation of DNA had occurred in either F8 or F8-4, and suggest that the apparent difference is due to a recombination anomaly.     

The Effects of Temperature Shock on Transcription and Replication in Rhynchosciara americana (Diptera: Sciaridae)

The chromosomal response to temperature shock in Rhynchosciara americana is similar to that observed in other Diptera. After a 33 degrees C/90 min or a 36 degrees C/30 min shock the reaction for RNA polymerase II (RpII) is enhanced at five loci. The most prominent of these was identified by in situ hybridization as the site of the hsp70 gene. At 33 degrees C, an accumulation of heat shock factor (HSF) and an increase in the level of RpII was observed at some heat shock loci after 5 min and reached a maximum after 15 min at most loci. The pattern of accumulation of HSF and RpII at individual heat shock loci was similar and their increases were generally coordinated among the loci. RpII gradually decreased at sites active prior to shock, the rate of decrease varying with the site. The B2 DNA puff retained RpII for a significant length of time while the histone locus still contained RpII after a shock of 90 min. With a 36 degrees C/30 min shock, the size of the heat shock puffs and the intensities of HSF and RpII peaked at 1-4 h post stress. The level of HSF declined rapidly after 1 h while the level of RpII remained high for an additional 4 h. The reaction of the DNA puffs to heat shock varied. Usually they did not regress completely and retained traces of RpII. BrdU incorporation continued at both amplifying and non-amplifying bands after shock but on average it appeared depressed for about 24 h post stress.     

Replication and recombination functions associated with the yeast plasmid, 2μ circle

By examining both the transformation efficiency of yeast of various plasmids containing defined regions of the 2μ circle genome and the characteristics of the resultant transformants, we have identified several regions of the 2μ circle genome which are involved in 2μ circle replication or recombination. First, by identifying those DNA fragments from the molecule which promote high frequency transformation of yeast, we have localized the origin of replication to a sequence partially within the large unique region, which, as determined by subsequent deletion analysis, extends from the middle of the inverted repeat region into the contiguous unique region. Second, by examining the relative efficiency of replication in yeast of hybrid plasmids containing either the entire 2μ circle genome or a fragment of 2μ circle encompassing the origin of replication, we have determined that efficient use of the 2μ circle origin requires some function or functions encoded in the molecule at a site away from the origin. Third, by examining the ability of a mutant 2μ circle molecule to undergo intramolecular recombination in yeast, we have identified a 2μ circle gene which codes for a product required for this process.