Why Are There So Many Diverse Replication Machineries?

The replicon model has initiated a major research line in molecular biology: the study of DNA replication mechanisms. Until now, the majority of studies have focused on a limited set of model organisms, mainly from Bacteria or Opisthokont eukaryotes (human, yeasts) and a few viral systems. However, molecular evolutionists have shown that the living world is more complex and diverse than believed when the operon model was proposed. Comparison of DNA replication proteins in the three domains, Archaea, Bacteria, and Eukarya, have surprisingly revealed the existence of two distinct sets of non-homologous cellular DNA replication proteins, one in Bacteria and the other in Archaea and Eukarya, suggesting that the last universal common ancestor possibly still had an RNA genome. A major puzzle is the presence in eukaryotes of the unfaithful DNA polymerase alpha (Pol α) to prime Okazaki fragments. Interestingly, Pol α is specifically involved in telomere biosynthesis, and its absence in Archaea correlates with the absence of telomeres. The recent discovery of telomere-like GC quartets in eukaryotic replication origins suggests a link between Pol α and the overall organization of the eukaryotic chromosome. As previously proposed by Takemura, Pol α might have originated from a mobile element of viral origin that played a critical role in the emergence of the complex eukaryotic genomes. Notably, most large DNA viruses encode DNA replication proteins very divergent from their cellular counterparts. The diversity of viral replication machineries compared to cellular ones suggests that DNA and DNA replication mechanisms first originated and diversified in the ancient virosphere, possibly explaining why they are so many different types of replication machinerie.     

Structure and function of SV40 large-T antigen

The small eukaryotic DNA tumour virus, SV40, has long provided a very useful model for the study of eukaryotic DNA replication and cellular transformation. The viral gene product, large-tumour (large-T) antigen, is essential for the initiation of viral DNA replication and the initiation and maintenance of SV40-virus-mediated cellular transformation. The large-T antigen is a complex multifunctional protein, and to delineate its activity more precisely in viral DNA replication and cellular transformation, small functional domains of the protein have been expressed in Escherichia coli and analysed by using a very extensive library of anti-T monoclonal antibodies.     

The eukaryotic replication complex and its affinity modification analysis

Replication of eukaryotic DNA is driven by a protein complex, in which the central part is played by DNA polymerases. Synthesis with eukaryotic DNA polymerases alpha, delta, and epsilon involves various replication factors, including the replication protein A, replication factor C, proliferating cell nuclear antigen, etc. Replication enzymes and factors also participate in DNA repair, which is in an interplay with DNA replication. The function of the entire multicomponent system is regulated by protein--nucleic acid and protein--protein interactions. The eukaryotic replication complex was not isolated as a stable supramolecular structure, suggesting its dynamic organization. Hence X-ray analysis and other instrumental techniques are hardly suitable for studying this system. An alternative approach is affinity modification. Its most promising version involves in situ generation of photoreactive DNA replication intermediates. The review considers the recent progress in photoaffinity modification studies of DNA polymerases, eukaryotic replication factors, and their interactions with DNA replication intermediates.     

Mechanisms and regulation of DNA replication initiation in eukaryotes

Cellular DNA replication is initiated through the action of multiprotein complexes that recognize replication start sites in the chromosome (termed origins) and facilitate duplex DNA melting within these regions. In a typical cell cycle, initiation occurs only once per origin and each round of replication is tightly coupled to cell division. To avoid aberrant origin firing and re-replication, eukaryotes tightly regulate two events in the initiation process: loading of the replicative helicase, MCM2-7, onto chromatin by the origin recognition complex (ORC), and subsequent activation of the helicase by its incorporation into a complex known as the CMG. Recent work has begun to reveal the details of an orchestrated and sequential exchange of initiation factors on DNA that give rise to a replication-competent complex, the replisome. Here, we review the molecular mechanisms that underpin eukaryotic DNA replication initiation – from selecting replication start sites to replicative helicase loading and activation – and describe how these events are often distinctly regulated across different eukaryotic model organisms.     

The DNA-A component of a plant geminivirus (Indian mung bean yellow mosaic virus) replicates in budding yeast cells

Understanding the biochemistry of DNA replication of the plant DNA viruses is important for the development of antiviral strategies. Since DNA replication is little studied in plants, a genetically tractable, easily culturable, eukaryotic model system is required to pursue such studies in a facile manner. Here we report the development of a yeast model system that supports DNA replication of a chosen geminivirus strain, Indian mung bean yellow mosaic virus. The replication of plasmid DNA in the model system relies specifically on the virus-derived elements and factors. Usage of this model system revealed the role of at least one hitherto unknown viral factor for viral DNA replication. The episomal characteristic of single-strandedness of replicated plasmid DNA was shown, and the expression of viral genes was also confirmed. This model system is expected to shed light on the machinery and mechanism involved in geminiviral DNA replication in plants.     

A rotary pumping model for helicase function of MCM proteins at a distance from replication forks

We propose an integrated model for eukaryotic DNA replication to explain the following problems: (1) How is DNA spooled through fixed sites of replication? (2) What and where are the helicases that unwind replicating DNA? (3) Why are the best candidates for replicative helicases, namely mini-chromosome maintenance (MCM) proteins, not concentrated at the replication fork? (4) How do MCM proteins spread away from loading sites at origins of replication? We draw on recent discoveries to argue that the MCM hexameric ring is a rotary motor that pumps DNA along its helical axis by simple rotation, such that the movement resembles that of a threaded bolt through a nut, and we propose that MCM proteins act at a distance from the replication fork to unwind DNA. This model would place DNA replication in a growing list of processes, such as recombination and virus packaging, that are mediated by ring-shaped ATPases pumping DNA by helical rotation.     

Plasmodium falciparum origin recognition complex subunit 5: functional characterization and role in DNA replication foci formation

The mechanism of DNA replication initiation and progression is poorly understood in the parasites, including human malaria parasite Plasmodium falciparum . Using bioinformatics tools and yeast complementation assay, we identified a putative homologue of Saccharomyces cerevisiae o rigin r ecognition c omplex subunit 5 in P. falciparum (PfORC5). PfORC5 forms distinct nuclear foci colocalized with the replication foci marker proliferating cell nuclear antigen (PfPCNA) and co-immunoprecipitates with PCNA during early-to-mid trophozoite stage replicating parasites. Interestingly, these proteins separate from each other at the non-replicating late schizont stage, citing the evidence of the presence of both PCNA and ORC components in replication foci during eukaryotic DNA replication. PfORC1, another ORC subunit, colocalizes with PfPCNA and PfORC5 at the beginning of DNA replication, but gets degraded at the late schizont stage, ensuring the regulation of DNA replication in the parasites. Further, we have identified putative PCNA-interacting protein box in PfORC1 that may explain in part the colocalization of PfORC and PfPCNA. Additionally, use of specific DNA replication inhibitor hydroxyurea affects ORC5/PCNA foci formation and parasitic growth. These results strongly favour replication factory model in the parasites and confer great potential to understand the co-ordination between ORC and PCNA during eukaryotic DNA replication in general.     

Is the nuclear matrix the site of DNA replication in eukaryotic cells?

Four types of experiment were carried out to test the recently proposed model of matrix-bound replication in eukaryotic cells. In experiments with pulse-labelling we found preferential association of newly replicated DNA with the matrix only when the procedure for isolation includes first high-salt treatment of isolated nuclei and then digestion with nucleases, or when prior to digestion the nuclei have been stored for a prolonged time. In both cases, however, evidence was found that this preferential association is due to a secondary, artifactual binding of the newly replicated chromatin region to the matrix elements. Pulse-chase experiments and experiments with continuous labelling were carried out to answer the question whether during replication the DNA is reeled through the replication complexes, i.e., whether newly replicated DNA is temporarily or permanently associated with the matrix. The results showed that at that time the matrix DNA does not move from its site of attachment. Since, according to the model of matrix-bound replication, the forks are assumed to be firmly anchored to high-salt resistant proteinaceous matrix structures, the chromatin fragments isolated with endonuclease not recognizing newly replicated DNA and purified by sucrose gradient centrifugation should be free of replication intermediates. The electronmicroscopic analysis of such fragments revealed the existence of intact replication micro-bubbles. Moreover, the fragments with replication configurations appeared as smooth chromatin fibres not attached to elements characteristic for the matrix. All these experiments suggest that the nuclear skeleton is not a native site of DNA replication in eukaryotic cells.     

GINS is a DNA polymerase epsilon accessory factor during chromosomal DNA replication in budding yeast

GINS is a protein complex found in eukaryotic cells that is composed of Sld5p, Psf1p, Psf2p, and Psf3p. GINS polypeptides are highly conserved in eukaryotes, and the GINS complex is required for chromosomal DNA replication in yeasts and Xenopus egg. This study reports purification and biochemical characterization of GINS from Saccharomyces cerevisiae. The results presented here demonstrate that GINS forms a 1:1 complex with DNA polymerase epsilon (Pol epsilon) holoenzyme and greatly stimulates its catalytic activity in vitro. In the presence of GINS, Pol epsilon is more processive and dissociates more readily from replicated DNA, while under identical conditions, proliferating cell nuclear antigen slightly stimulates Pol epsilon in vitro. These results strongly suggest that GINS is a Pol epsilon accessory protein during chromosomal DNA replication in budding yeast. Based on these results, we propose a model for molecular dynamics at eukaryotic chromosomal replication fork.     

Eukaryotic DNA replication.

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.     

DNA polymerase epsilon: in search of a function.

The current model of eukaryotic DNA replication involves the two DNA polymerases delta and alpha as the leading and lagging strand enzymes, respectively. A DNA polymerase first discovered in yeast has now been found in all eukaryotic cells and is termed DNA polymerase epsilon. In yeast, the gene for DNA polymerase epsilon has recently been found to be essential for viability, raising new questions about its functions.     

A double-loop model for the replication of eukaryotic DNA

Coordinated DNA synthesis of both strands at the replication fork by a fixed 'replisome' may cause dynamic and topological problems. Based upon known properties of DNA helicase, DNA primase and DNA topoisomerases, and on novel properties of DNA polymerases and DNA ligase, we propose a 'double-loop' model for the replication of eukaryotic DNA that could minimize such problems.     

Le modèle du réplicon est-il applicable aux eucaryotes supérieurs?

Thirty-five years ago, the Replicon model was proposed by Jacob, Brenner and Cuzin to explain the regulation of the Escherichia coli DNA replication. In this model, a genetic element, the replicator, would function as a target for a positive-acting initiator protein to drive the initiation of replication. This simple idea has been extremely useful in providing a framework to explain how the initiation of DNA replication occurs in all organisms. The identification of autonomously replicating sequences (ARSs) in budding yeast was the first extension of the Replicon model to eukaryotic chromosomes. In the higher eukaryotes, many biochemically defined replication start sites have been identified; nevertheless there is little genetic data indicating that these sites contain DNA sequences that are essential for replication. Moreover, in early Xenopus or Drosophila embryos, specific DNA sequences are not required either for initiating DNA replication or for preventing rereplication within a single cell cycle. This apparently fundamental difference between replicators in yeast and metazoan embryos may be more superficial than initially thought. In fact, during the past several years, an eukaryotic initiator conserved from yeast to man and also present in embryonic cells, the origin recognition complex (ORC), has been characterized, suggesting that the initiation mechanism should be essentially the same in prokaryotes and eukaryotes. In addition, the efficient once-per-cell-cycle replication of DNA is ensured in eukaryotes by a simple two-step mechanism in which the assembly of stable prereplicative complexes (PreRCs) at origins precedes and is temporally separated from the firing of these origins. Regulation of this process by cyclin-dependent kinases ensures that when origins fire, the cell is no longer competent to form new PreRCs. Now, it is important to understand how these complexes are remodeled or disassembled during replication initiation to trigger the transition from a stable origin-bound complex to a mobile replication machine.     

Control of SV40 DNA replication by protein phosphorylation: a model for cellular DNA replication?

SV40 DNA replication has been studied extensively as a model for eukaryotic DNA replication. The initiation of SV40 DNA replication depends on certain cellular enzymes and on a multifunctional viral phosphoprotein, T antigen, whose activity is controlled positively and negatively by its phosphorylation state. Several cellular protein kinases and phosphatases that act on T antigen have now been identified. The recent elucidation of the step in initiation that is sensitive to T antigen's phosphorylation state raises the question of whether initiation of cellular DNA replication may utilize a similar regulatory mechanism.     

Replication origins in yeast chromosomes

DNA replication initiates at many sites in eukaryotic chromosomes. It has been difficult to isolate such replication origins, but a family of sequences from the yeast genome have properties which suggest that they may serve this function. The identification of these sequences together with sophisticated methods of genetic analysis, make yeast a useful organism for the study of eukaryotic DNA replication.     

Yeast replicative DNA polymerases and their role at the replication fork.

The budding yeast, Saccharomyces cerevisiae, is an excellent model system for the study of DNA polymerases and their roles in DNA replication, repair, and recombination. Presently ten DNA polymerases have been purified and characterized from S. cerevisiae. Rapid advances in genome sequencing projects for yeast and other organisms have greatly facilitated and accelerated the identification of yeast enzymes and their homologues in other eukaryotic species. This article reviews current available research on yeast DNA polymerases and their functional roles in DNA metabolism. Relevant information about eukaryotic homologues of these enzymes will also be discussed.     

Eukaryotic DNA Replication

Abstract

The past decade has witnessed an exciting evolution in our understanding of eukaryotic DNA replication at the molecular level. Progress has been particularly rapid within the last few years due to the convergence of research on a variety of cell types, from yeast to human, encompassing disciplines ranging from clinical immunology to the molecular biology of viruses. New eukaryotic DNA replicases and accessory proteins have been purified and characterized, and some have been cloned and sequenced. In vitro systems for the replication of viral DNA have been developed, allowing the identification and purification of several mammalian replication proteins. In this review we focus on DNA polymerases alpha and delta and the polymerase accessory proteins, their physical and functional properties, as well as their roles in eukaryotic DNA replication.     

DNA polymerase delta: a second eukaryotic DNA replicase

During the past few years significant progress has been made in our understanding of the structure and function of the proteins involved in eukaryotic DNA replication. Data from several laboratories suggest that, in contrast to prokaryotic DNA replication, two distinct DNA polymerases are required for eukaryotic DNA replication, i.e. DNA polymerase delta for the synthesis of the leading strand and DNA polymerase alpha for the lagging strand. Several accessory proteins analogous to prokaryotic replication factors have been identified and some of these are specific for pol delta whereas others affect both DNA replicases. The replicases and their accessory proteins appear to be highly conserved in eukaryotes, as homologous proteins have been found in species ranging from humans to yeast.     

Changes in the subcellular localization of replication initiation proteins and cell cycle proteins during G1- to S-phase transition in mammalian cells

DNA replication in eukaryotic cells is restricted to the S-phase of the cell cycle. In a cell-free replication model system, using SV40 origin-containing DNA, extracts from G1 cells are inefficient in supporting DNA replication. We have undertaken a detailed analysis of the subcellular localization of replication proteins and cell cycle regulators to determine when these proteins are present in the nucleus and therefore available for DNA replication. Cyclin A and cdk2 have been implicated in regulating DNA replication, and may be responsible for activating components of the DNA replication mitiation complex on entry into S-phase. G1 cell extracts used for in vitro replication contain the replication proteins RPA (the eukaryotic single-stranded DNA binding protein) and DNA polymerase as well as cdk2, but lack cyclin A. On localizing these components in G1 cells we find that both RPA and DNA polymerase are present as nuclear proteins, while cdk2 is primarily cytoplasmic and there is no detectable cyclin A. An apparent change in the distribution of these proteins occurs as the cell enters S-phase. Cyclin A becomes abundant and both cyclin A and cdk2 become localized to the nucleus in S-phase. In contrast, the RPA-34 and RPA-70 subunits of RPA, which are already nuclear, undergo a transition from the uniform nuclear distribution observed during G1, and now display a distinct punctate nuclear pattern. The initiation of DNA replication therefore most likely occurs by modification and activation of these replication initiation proteins rather than by their recruitment to the nuclear compartment.