Species-specific in vitro synthesis of DNA containing the polyoma virus origin of replication.

In vitro replication of DNA containing the polyoma (Py) virus origin of replication has been carried out with cell-free extracts prepared from mouse FM3A cells. The in vitro system required the Py virus-encoded large tumor (T) antigen, DNA containing the Py virus origin of replication, ATP, and an ATP-regenerating system. The replication reaction was inhibited by aphidicolin, suggesting the involvement of DNA polymerase alpha in this system. Simian virus 40 (SV40) T antigen could not substitute for the Py T antigen. Cell extracts prepared from HeLa cells, a source that replicates SV40 DNA in the presence of SV40 T antigen, replicated Py DNA poorly. The addition of purified DNA polymerase alpha-primase complex isolated from FM3A cells enabled HeLa cell extracts to replicate Py DNA with the same efficiency as FM3A cell extracts. Complementary experiments have shown that FM3A cell extracts do not support SV40 DNA replication unless supplemented with DNA polymerase alpha-primase complex from HeLa cells [Murakami, Y., Wobbe, C.R., Weissbach, L., Dean, F.B. & Hurwitz, J. (1986) Proc. Natl. Acad. Sci. USA 83, 2869-2873]. These results indicate that the host-cell source of the DNA polymerase alpha-primase complex plays an important role in discriminating between SV40 T antigen- and Py T antigen-dependent replication of their homologous DNA in vitro. This may explain the host-range specificity of these viruses in vivo.     

Replication of simian virus 40 origin-containing DNA in vitro with purified proteins.

Simian virus 40 (SV40) DNA replication dependent on the SV40 origin of replication and the SV40 large tumor (T) antigen has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, these components included the DNA polymerase alpha-primase complex, topoisomerase I, and a fraction that contained a single-stranded DNA binding protein. The latter protein, which sediments at 5.1 S on glycerol gradients and copurifies with two major protein species of 72 and 76 kDa, was isolated solely by its ability to support SV40 DNA replication. The purified system retained the species-specific DNA polymerase alpha-primase requirement previously observed with crude fractions; the complex from HeLa cells supported SV40 replication, whereas that from calf thymus and mouse cells did not. DNA containing the polyomavirus origin of replication was replicated in a system containing polyomavirus T antigen, the HeLa single-stranded DNA binding protein-containing fraction, and DNA polymerase alpha-primase complex from mouse, but not HeLa, cells. While crude fractions yielded closed circular duplex DNA, none was detected with the purified system. Nevertheless, the addition of a crude fraction to the purified system yielded closed circular monomer products.     

Fidelity of a human cell DNA replication complex.

We have measured the fidelity of bidirectional, semiconservative DNA synthesis by a human DNA replication complex in vitro. Replication was performed by extracts of HeLa cells in the presence of simian virus 40 (SV40) large tumor antigen by using a double-stranded phage M13mp2 DNA template containing the SV40 origin of replication and either of two different target sequences for scoring mutations in the lacZ alpha-complementation gene, which encodes the alpha region (specifying the amino-terminal portion) of beta-galactosidase. Replicative synthesis was substantially more accurate than synthesis by the human DNA polymerase alpha-DNA primase complex purified from HeLa cell extracts by immunoaffinity chromatography, suggesting that additional factors or activities in the extract may increase fidelity during bidirectional replication. However, by using a sensitive opal codon reversion assay, single-base substitution errors were readily detected in the replication products at frequencies significantly higher than estimated spontaneous mutation rates in vivo. These data suggest that additional fidelity factors may be present during chromosomal replication in vivo and/or that the fidelity of replication alone does not account for the low spontaneous mutation rates in eukaryotes.     

Studies on the initiation and elongation reactions in the simian virus 40 DNA replication system.

The synthesis of oligoribonucleotides by DNA primase in the presence of duplex DNA containing the simian virus 40 (SV40) origin of replication was examined. Small RNA chains (10-15 nucleotides) were synthesized in the presence of the four common ribonucleoside triphosphates, SV40 large tumor antigen (T antigen), the human DNA polymerase alpha (pol alpha)-DNA primase complex, the human single-stranded DNA-binding protein (HSSB), and topoisomerase I isolated from HeLa cells. The DNA primase-catalyzed reaction showed an absolute requirement for T antigen, HSSB, and pol alpha. The requirement for HSSB was not satisfied by other SSBs that can support the T-antigen-catalyzed unwinding of DNA containing the SV40 origin of replication. Oligoribonucleotide synthesis occurred with a lag that paralleled the lag observed in DNA synthesis. These results indicate that the specificity for the HSSB in the SV40 replication reaction is due to the pol alpha-primase-mediated synthesis of the Okazaki fragments. In contrast to this specificity, the elongation of Okazaki fragments can be catalyzed by a variety of different DNA polymerases, including high levels of pol alpha, the polymerase delta holoenzyme, T4 polymerase holoenzyme, the Escherichia coli polymerase III holoenzyme, and other polymerases. These observations suggest that leading-strand synthesis in the in vitro SV40 replication system can be nonspecific.     

Roles of DNA topoisomerases in simian virus 40 DNA replication in vitro.

We examined the roles of DNA topoisomerases in the replication of simian virus 40 (SV40) DNA in a cell-free system composed of an extract from HeLa cells supplemented with purified SV40 tumor antigen. When the activities of both topoisomerase I (EC 5.99.1.2) and topoisomerase II (EC 5.99.1.3) in the extract were blocked with specific inhibitors or antibodies, DNA synthesis was decreased by a factor of 15-20. Addition of purified HeLa DNA topoisomerase II to extracts immunologically depleted of both topoisomerases completely restored replication, and the replication products consisted largely of monomeric daughter molecules. Addition of purified HeLa DNA topoisomerase I to depleted extracts restored DNA synthesis, but the primary products were multiply intertwined, catenated daughter molecules. We conclude that DNA topoisomerases have at least two roles in the replication of SV40 DNA. Either topoisomerase I or topoisomerase II is sufficient to provide the unlinking activity necessary for fork propagation during SV40 DNA replication. However, topoisomerase II is uniquely required for the segregation of newly synthesized daughter molecules.     

Synthesis of DNA containing the simian virus 40 origin of replication by the combined action of DNA polymerases alpha and delta.

Proliferating-cell nuclear antigen (PCNA) mediates the replication of simian virus 40 (SV40) DNA by reversing the effects of a protein that inhibits the elongation reaction. Two other protein fractions, activator I and activator II, were also shown to play important roles in this process. We report that activator II isolated from HeLa cell extracts is a PCNA-dependent DNA polymerase delta that is required for efficient replication of DNA containing the SV40 origin of replication. PCNA-dependent DNA polymerase delta on a DNA singly primed phi X174 single-stranded circular DNA template required PCNA, a complex of the elongation inhibitor and activator I, and the single-stranded DNA-binding protein essential for SV40 DNA replication. DNA polymerase delta, in contrast to DNA polymerase alpha, hardly used RNA-primed DNA templates. These results indicate that both DNA polymerase alpha and delta are involved in SV40 DNA replication in vitro and their activity depends on PCNA, the elongation inhibitor, and activator I.     

Purification of replication protein C, a cellular protein involved in the initial stages of simian virus 40 DNA replication in vitro.

The replication of simian virus 40 (SV40) DNA is dependent upon a single viral protein [tumor (T) antigen] and multiple cellular proteins. To define the required cellular proteins, we have made use of a cell-free system that supports the replication of plasmid DNA molecules containing the SV40 origin of replication. We report here the purification from HeLa cell extracts of replication protein C (RP-C), a previously undescribed protein that is required to reconstitute efficient DNA replication in vitro. Highly purified preparations of RP-C contain two closely related polypeptides of 32 and 34 kDa. Preincubation of purified RP-C with T antigen and the DNA template largely eliminates the delay normally observed before the onset of rapid DNA synthesis. In addition, RP-C stimulates the unwinding of duplex DNA molecules containing the SV40 replication origin in a reaction that requires T antigen and a single-stranded DNA binding protein. These observations suggest that RP-C is involved in the initial steps of SV40 DNA replication in vitro.     

Simian virus 40 DNA replication in vitro.

Soluble extracts prepared from monkey cells (COS-1 or BSC-40) infected with simian virus 40 (SV40) catalyze the efficient replication of exogenously added plasmid DNA molecules containing the cloned SV40 origin of replication. Extracts prepared from uninfected monkey cells also support origin-dependent replication in vitro but only in the presence of added SV40 large tumor (T) antigen. Very little DNA synthesis is observed when the cloned viral origin contains a 4-base-pair deletion mutation known to abolish SV40 DNA replication in vivo or when the parental plasmid vector lacking SV40 sequences is employed as template. The in vitro replication reaction proceeds via branched intermediates (theta structures) that resemble in vivo replication intermediates. Replication is sensitive to aphidicolin but relatively resistant to dideoxythymidine triphosphate. The product of the reaction consists of covalently closed circular DNA molecules that contain full-length daughter strands hydrogen bonded to the parental template. These observations support the conclusion that replication in the in vitro system closely resembles SV40 DNA replication in vivo. The system provides a biochemical assay for the replication activity of SV40 T antigen and should also facilitate the purification and functional characterization of cellular proteins involved in DNA replication.     

Conservation of structure and function of DNA replication protein A in the trypanosomatid Crithidia fasciculata.

Human replication protein A (RP-A) is a three-subunit protein that is required for simian virus 40 (SV40) replication in vitro. The trypanosome homologue of RP-A has been purified from Crithidia fasciculata. It is a 1:1:1 complex of three polypeptides of 51, 28, and 14 kDa, binds single-stranded DNA via the large subunit, and is localized within the nucleus. C. fasciculata RP-A substitutes for human RP-A in the large tumor antigen-dependent unwinding of the SV40 origin of replication and stimulates both DNA synthesis and DNA priming by human DNA polymerase alpha/primase, but it does not support efficient SV40 DNA replication in vitro. This extraordinary conservation of structure and function between human and trypanosome RP-A suggests that the mechanism of DNA replication, at both the initiation and the elongation level, is conserved in organisms that diverged from the main eukaryotic lineage very early in evolution.     

Adenovirus DNA replication in vitro: synthesis of full-length DNA with purified proteins.

A protein required for the elongation of replicating intermediates of adenovirus (Ad) DNA to full length has been isolated and characterized. This factor, isolated from nuclear extracts of uninfected HeLa cells, has been designated nuclear factor II. In the presence of Ad DNA with proteins at each 5' end (Ad DNA-protein) and three proteins coded for by the Ad genome [the preterminal protein (pTP), the DNA polymerase (Ad Pol), and the DNA binding protein (Ad DBP)], nuclear factor II complementing activity is detected only in the presence of host nuclear factor I. Highly purified preparations of nuclear factor II that are free of detectable DNA polymerase alpha, beta, and gamma activities contain a DNA topoisomerase activity. Furthermore, type I DNA topoisomerases purified from HeLa cells and calf thymus substitute for nuclear factor II complementing activity in the in vitro Ad DNA replication system. These results indicate that a protein that is involved in higher order DNA structure is required for Ad replication. This protein plus the purified proteins described above carry out the initiation and synthesis of full-length 36,000-base-pair Ad DNA.     

Construction and analysis of simian virus 40 origins defective in tumor antigen binding and DNA replication.

We have inserted a 311-base pair DNA fragment containing the simian virus 40 (SV40) origin of DNA replication, the early promoter, and the tumor (T) antigen binding sites into a bacterial plasmid and cloned it. This recombinant plasmid, pSV01, binds to a purified T antigen in vitro and replicates in monkey cells when supplied with large T antigen. A series of deletion mutations was generated in the origin sequences of pSV01 DNA by mutagenesis in vitro. The replication of these mutant DNAs in monkey cells was compared with their ability to bind to purified D2 protein. Mutant DNAs deficient in binding to D2 protein also exhibit reduced levels of replication in monkey cells. These findings provide biochemical evidence that the initiation of SV40 DNA synthesis may involve a direct interaction of T antigen with sequences at the origin of replication.     

Model system for DNA replication of a plasmid DNA containing the autonomously replicating sequence from Saccharomyces cerevisiae.

A negatively supercoiled plasmid DNA containing autonomously replicating sequence (ARS) 1 from Saccharomyces cerevisiae was replicated with the proteins required for simian virus 40 DNA replication. The proteins included simian virus 40 large tumor antigen as a DNA helicase, DNA polymerase alpha.primase, and the multisubunit human single-stranded DNA-binding protein from HeLa cells; DNA gyrase from Escherichia coli, which relaxes positive but not negative supercoils, was included as a "swivelase." DNA replication started from the ARS region, proceeded bidirectionally with the synthesis of leading and lagging strands, and resulted in the synthesis of up to 10% of the input DNA in 1 h. The addition of HeLa DNA topoisomerase I, which relaxes both positive and negative supercoils, to this system inhibited DNA replication, suggesting that negative supercoiling of the template DNA is required for initiation. These results suggest that DNA replication starts from the ARS region where the DNA duplex is unwound by torsional stress; this unwound region can be recognized by a DNA helicase with the assistance of the multisubunit human single-stranded DNA-binding protein.     

Studies on the DNA elongation inhibitor and its proliferating cell nuclear antigen-dependent control in simian virus 40 DNA replication in vitro.

A 120-kDa protein that blocks DNA termini has been purified from extracts of HeLa cells. This protein inhibits the action of a number of enzymes that catalyze reactions involving the 5' and 3' ends of DNA (DNA ligase, 3' and 5' exonucleases, and DNA polymerase alpha). The 120-kDa protein blocks the synthesis of long DNA chains that are normally formed during simian virus 40 DNA replication, causing the accumulation of small DNA fragments. The effects of this protein can be reversed by the addition of proliferating cell nuclear antigen and other protein fractions (activators).     

An inhibitor of the in vitro elongation reaction of simian virus 40 DNA replication is overcome by proliferating-cell nuclear antigen.

The replication of simian virus 40 (SV40) origin-containing DNA has been reconstituted by using SV40 large tumor (T) antigen and cellular proteins purified from HeLa cells. This replication reaction is unaffected by proliferating-cell nuclear antigen (PCNA). In contrast, PCNA has been reported to stimulate SV40 DNA synthesis carried out with crude fractions [Prelich, G., Kostura, M., Marshak, D. R., Mathews, M. B. & Stillman, B. (1987) Nature (London) 326, 471-475]. This difference is caused by the presence of a protein in crude fractions that inhibits the elongation of nascent DNA chains during replication. In the presence of PCNA, crude fractions containing this elongation inhibition factor can extend DNA chains. We describe the partial purification of this inhibitor and show that its addition limited SV40 DNA replication to the synthesis of short chains, an effect reversed by the addition of PCNA. However, the reversal of the inhibition by PCNA in the SV40 system required additional protein fractions distinct from PCNA and the enzymes constituting the purified system. These results suggest that the PCNA-mediated effect on SV40 DNA replication may be indirect. Such an interplay between negative and positive regulatory functions including PCNA may contribute to the control of DNA synthesis characteristic of the eukaryotic cell cycle.     

Reconstitution of simian virus 40 DNA replication with purified proteins.

Replication of plasmid DNA molecules containing the simian virus 40 (SV40) origin of DNA replication has been reconstituted with seven highly purified cellular proteins plus the SV40 large tumor (T) antigen. Initiation of DNA synthesis is absolutely dependent upon T antigen, replication protein A, and the DNA polymerase alpha-primase complex and is stimulated by the catalytic subunit of protein phosphatase 2A. Efficient elongation of nascent chains additionally requires proliferating cell nuclear antigen, replication factor C, DNA topoisomerase I, and DNA polymerase delta. Electron microscopic studies indicate that DNA replication begins at the viral origin and proceeds via intermediates containing two forks that move in opposite directions. These findings indicate that the reconstituted replication reaction has many of the characteristics expected of authentic viral DNA replication.     

Requirement for two DNA polymerases in the replication of simian virus 40 DNA in vitro.

DNA polymerase alpha-primase has long been considered the primary, if not sole, replicative DNA polymerase in eukaryotic cells. However, recent experiments have provided indirect evidence that a second DNA polymerase may play a role in DNA replication. To identify cellular proteins necessary for DNA synthesis in mammalian cells, we have been studying the cell-free system developed for the replication of simian virus 40 DNA. In this report, we present direct evidence that a second DNA polymerase is required in addition to DNA polymerase alpha-primase complex to obtain efficient replication of simian virus 40 origin-containing DNA. This DNA polymerase activity is not affected by monoclonal antibodies that inhibit the activity of DNA polymerase alpha and is relatively resistant to the inhibitor [N2-(p-n-butylphenyl)-9-(2-deoxy-beta-D-ribofuranosyl)guanine 5'-triphosphate]. Moreover, the activity of the polymerase is highly dependent upon the accessory protein, proliferating-cell nuclear antigen. These characteristics are consistent with the hypothesis that this second DNA polymerase is DNA polymerase delta.     

Preferential replication of repeated DNA sequences in nuclei isolated from soybean cells grown in suspension culture.

Nuclei isolated from suspension cultures of soybean cells are able to replicate DNA in vitro for short periods of time. The reaction is not blocked by dideoxythymidine triphosphate, suggesting that DNA polymerase alpha is involved. Reassociation kinetics, restriction analysis, and isopycnic centrifugation of the DNA synthesized in vitro show that repeated sequences are preferentially replicated in isolated nuclei. However, some repeated sequences are not replicated in vitro, including those coding for ribosomal RNA. The addition of cytoplasmic or yeast extracts clearly stimulates DNA replication in vitro but does not improve the ability of nuclei to replicate single copy DNA. Incubation of cells with cytokinins prior to the isolation of nuclei modifies the pattern of replication of repeated sequences in vitro. These experiments suggest that the replication of single copy and repeated nuclear sequences have different requirements and may be controlled in a different way in plant cells.     

Replicative enzymes, DNA polymerase alpha (pol alpha), and in vitro ageing

Normal cells in culture are used to investigate the underlying mechanisms of DNA synthesis because they retain regulatory characteristics of the in vivo replication machinery. During the last few years new studies have identified a number of genetic changes that occur during in vitro ageing, providing insight into the progressive decline in biological function that occurs during ageing. Maintaining genomic integrity in eukaryotic organisms requires precisely coordinated replication of the genome during mitosis, which is the most fundamental aspect of living cells. To achieve this coordinated replication, eukaryotic cells employ an ordered series of steps to form several key protein assemblies at origins of replication. Major progress has recently been made in identifying the enzymes, and other proteins, of DNA replication that are recruited to origin sites and the order in which they are recruited during the process of replication. More than 20 proteins, including DNA polymerases, have been identified as essential components that must be preassembled at replication origins for the initiation of DNA synthesis. Of the polymerases, DNA polymerase alpha-primase (pol alpha) is of particular importance since its function is fundamental to understanding the initiation mechanism of eukaryotic DNA replication. DNA must be replicated with high fidelity to ensure the accurate transfer of genetic information to progeny cells, and decreases in DNA pol alpha activity and fidelity, which are coordinated with cell cycle progression, have been shown to be important facets of a probable intrinsic cause of genetic alterations during in vitro ageing. This has led to the proposal that pol alpha activity and function is one of the crucial determinants in ageing. In this review we summarize the current state of knowledge of DNA pol alpha function in the regulation of DNA replication and focus in particular on its interactive tasks with other proteins during in vitro ageing.     

DNA supercoiling of recombinant plasmids in mammalian cells.

We have used chloroquine/agarose gel electrophoresis and a blot-hybridization technique to study the modulation of superhelicity of extrachromosomal DNA in mammalian cells. The high sensitivity of the procedure has allowed us to measure the change in the specific linking difference or superhelical density (sigma) of a plasmid, psvo alpha 1p3d, after its introduction into COS-7 cells by DNA transfection. Because the molecular weight of psvo alpha 1p3d is approximately the same as that of simian virus 40 (SV40) DNA, the latter can be used as a standard for estimating the average linking difference or number of superhelical turns (tau) of psvo alpha 1p3d after separation of the different supercoiled species on chloroquine/agarose gels. It was found that transfection of monkey cells with either fully supercoiled psvo alpha 1p3d isolated from bacteria (tau = -27 +/- 1, sigma congruent to -0.051) or its relaxed form after treatment with DNA topoisomerase I yields psvo alpha 1p3d samples of the same tau and sigma values of -20 +/- 1 and -0.038, respectively. The difference between the tau values of psvo alpha 1p3d and SV40 in COS-7 cells, in which both plasmids undergo rounds of replication, corresponds to an average difference of 5 +/- 1 superhelical turns. Plasmid psvo alpha 1p3d remains at this lower level of superhelicity for at least 72 hr. The distribution in linking numbers of the topoisomers of psvo alpha 1p3d isolated from transfected COS cells is also more heterogeneous than that of SV40 DNA. These results suggest that the regulation of DNA supercoiling and chromatin assembly may be closely associated with specific DNA sequences. The approach presented here should have a wide application in the study of the regulation and functional role(s) of DNA supercoiling of plasmids in mammalian cells.