In vitro replication of DNA containing either the SV40 or the polyoma origin

The replication of DNA containing either the polyoma or SV40 origin has been done in vitro. Each system requires its cognate large-tumour antigen (T antigen) and extracts from cells that support its replication in vivo. The host-cell source of DNA polymerase alpha - primase complex plays an important role in discriminating between polyoma T antigen and SV40 T antigen-dependent replication of their homologous DNA. The SV40 origin- and T antigen-dependent DNA replication has been reconstituted in vitro with purified protein components isolated from HeLa cells. In addition to SV40 T antigen, HeLa DNA polymerase alpha - primase complex, eukaryotic topoisomerase I and a single-strand DNA binding protein from HeLa cells are required. The latter activity, isolated solely by its ability to support SV40 DNA replication, sediments and copurifies with two major protein species of 72 and 76 kDa. Although crude fractions yielded closed circular monomer products, the purified system does not. However, the addition of crude fractions to the purified system resulted in the formation of replicative form I (RFI) products. We have separated the replication reaction with purified components into multiple steps. In an early step, T antigen in conjunction with a eukaryotic topoisomerase (or DNA gyrase) and a DNA binding protein, catalyses the conversion of a circular duplex DNA molecule containing the SV40 origin to a highly underwound covalently closed circle. This reaction requires the action of a helicase activity and the SV40 T antigen preparation contains such an activity. The T antigen associated ability to unwind DNA copurified with other activities intrinsic to T antigen (ability to support replication of SV40 DNA containing the SV40 origin, poly dT-stimulated ATPase activity and DNA helicase).     

Differential increase in topoisomerase II in simian virus 40-infected cells.

The time course of expression of topoisomerase I, topoisomerase II, and simian virus 40 (SV40) large tumor (T) antigen was determined in whole-cell extracts of uninfected versus SV40-infected TC7 cells. After a minor increase, the level of topoisomerase I remained fairly constant throughout the time course in both uninfected and SV40-infected cells. In contrast, the level of topoisomerase II increased markedly in SV40-infected cells but not in uninfected cells following the appearance of SV40 T antigen.     

Initiation of simian virus 40 DNA replication requires the interaction of a specific domain of human DNA polymerase alpha with large T antigen.

Initiation of cell-free simian virus 40 (SV40) DNA replication requires the interaction of DNA polymerase alpha/primase with a preinitiation complex containing the viral T antigen and cellular proteins, replication protein A, and topoisomerase I or II. To further understand the molecular mechanisms of the transition from preinitiation to initiation, the intermolecular interaction between human DNA polymerase alpha and T antigen was investigated. We have demonstrated that the human DNA polymerase alpha catalytic polypeptide is able to associate with SV40 large T antigen directly under physiological conditions. A physical association between these two proteins was detected by coimmunoprecipitation with monoclonal antibodies from insect cells coinfected with recombinant baculoviruses. A domain of human polymerase alpha physically interacting with T antigen was identified within the amino-terminal region from residues 195 to 313. This domain of human polymerase alpha was able to form a nonproductive complex with T antigen, causing inhibition of the SV40 DNA replication in vitro. Kinetics of the inhibition indicated that this polymerase domain can inhibit viral replication only during the preinitiation stage. Extra molecules of T antigen could partially overcome the inhibition only prior to initiation complex formation. The data support the conclusion that initiation of SV40 DNA replication requires the physical interaction of T antigen in the preinitiation complex with the amino-terminal domain of human polymerase alpha from amino acid residues 195 to 313.     

Chromatin assembly during SV40 DNA replication in vitro

A cytosol extract from human 293 cells supports efficient replication of SV40 origin-containing plasmid DNA in the presence of the SV40 T antigen. Addition of a nuclear extract from the same cells promotes negative supercoiling of the replicated DNA but not the bulk of the unreplicated DNA. The level of superhelicity is affected by the concentrations of T antigen and nuclear extract factors and by the time of addition of the nuclear extract. The replicated DNA in isolated DNA-protein complexes resists relaxation by purified HeLa cell topoisomerase I. Micrococcal nuclease digestion, sucrose gradient sedimentation, and electron microscopy demonstrate that the negative supercoils result from assembly of the replicating DNA into a chromatin structure. These results suggest that, during DNA replication, the core histones can be assembled on both sides of the replication fork by an active, replication-linked mechanism that does not require a template of preexisting nucleosomes.     

Monoclonal antibody analysis of simian virus 40 small t-antigen expression in infected and transformed cells.

The monoclonal antibody PAb280 binds to small t antigen but not to large T antigen. Its binding site within the unique region of small t antigen was localized by studying its reaction with simian virus 40 mutants, other papovaviruses, and bacterial expression vectors coding for fragments of small t antigen. The antibody was used to define the cellular location of small t antigen by immunocytochemistry and by immunoprecipitation of subcellular extracts of infected cells. PAb280 reacts strongly with a cytoplasmic form of small t antigen that appears to be associated with the cytoskeleton and is not detected by antibodies directed to the common N terminus of small t and large T antigens. Immunoperoxidase staining of cells infected by the simian virus 40 defective strain SV402 with PAb280 and other anti-T antibodies demonstrated that this virus produced an N-terminal fragment of large T antigen as well as small t antigen. In cells infected by the virus, this fragment was located in the cell nucleus but was very unstable. These results suggest that the activity of the SV402 virus in transformation assays may not be entirely due to the action of small t antigen alone.     

A deletion in the simian virus 40 large T antigen impairs lytic replication in monkey cells in vivo but enhances DNA replication in vitro: new complementation function of T antigen.

We describe a new complementation function within the simian virus 40 (SV40) A gene. This function is required for viral DNA replication and virus production in vivo but, surprisingly, does not affect any of the intrinsic enzymatic functions of T antigen directly required for in vitro DNA replication. Other well-characterized SV40 T-antigen mutants, whether expressed stably from integrated genomes or in cotransfection experiments, complement these mutants for in vivo DNA replication and plaque formation. These new SV40 mutants were isolated and cloned from human cells which stably carry the viral DNA. The alteration in the large-T-antigen gene was shown by marker rescue and nucleotide sequence analysis to be a deletion of 322 bp spanning the splice-donor site of the first exon, creating a 14-amino-acid deletion in the large T antigen. The mutant gene was expressed in H293 human cells from an adenovirus vector, and the protein was purified by immunoaffinity chromatography. The mutant protein directs greater levels of DNA replication in vitro than does the wild-type protein. Moreover, the mutant protein reduces the lag time for in vitro DNA synthesis and can be diluted to lower levels than wild-type T antigen and still promote good replication, which is in clear contrast to the in vivo situation. These biochemical features of the protein are independent of the source of the cellular replication factors (i.e., HeLa, H293, COS 7, or CV1 cells) and the cells from which the T antigens were purified. The mutant T antigen does not transform Rat-2 cells. Several different models which might reconcile the differences observed in vivo and in vitro are outlined. We propose that the function of T antigen affected prepares cells for SV40 replication by activation of a limiting cellular replication factor. Furthermore, a link between the induction of a cellular replication factor and transformation by SV40 is discussed.     

Characterization of a camptothecin-resistant human DNA topoisomerase I in an in vitro system for Simian virus 40 DNA replication.

DNA topoisomerase I was required for bidirectional DNA replication in an in vitro system for Simian virus 40 (SV40) DNA replication with purified proteins in which the replication fork moved at the rate of 260 nucleotides/min on average. DNA topoisomerase I purified from camptothecin-resistant human lymphoblastoid cells, which confers high resistance of cellular DNA replication to camptothecin [Andoh, T., Ishii, K., Suzuki, Y., Ikegami, Y., Kusunoki, Y., Takemoto, Y. & Okada, K. (1987) Proc. Natl Acad. Sci. USA 84, 5565-5569], was characterized using this system. The activity of stimulating bidirectional DNA replication was comparable between two topoisomerase I from parental and resistant cells, i.e. in its dose-response relationship and in its time course for DNA synthesis. Camptothecin severely inhibited the leading as well as the lagging strand synthesis in the reaction containing the wild type topoisomerase I but not the mutant type topoisomerase I. The mutant type topoisomerase I was over 125-fold as resistant to camptothecin as the wild type topoisomerase I. These results are in good agreement with those on the sensitivity of cellular DNA synthesis to camptothecin in the resistant cells. These findings suggest that topoisomerase I is involved in cellular DNA replication as a swivelase and the mutation conferring camptothecin-resistance on the enzyme does not affect its functional efficiency in this system.     

Topoisomerase II plays an essential role as a swivelase in the late stage of SV40 chromosome replication in vitro.

The effects of topoisomerases I and II on the replication of SV40 DNA were examined using an in vitro replication system of purified proteins that constitutes the monopolymerase system. In the presence of the two topoisomerases, two distinct nascent DNAs were formed. One product arising from the replication of the leading template strand was approximately half the size of the template DNA, whereas the other product derived from the lagging template strand consisted of short DNAs. These products were synthesized from both SV40 naked DNA and SV40 chromosomes. For the replication of SV40 naked DNA, either topoisomerase I or II maintained replication fork movement and supported complete leading strand synthesis. When SV40 chromosomes were replicated with the same proteins, reactions containing only topoisomerase I produced shorter leading strands. However, mature size DNA products accumulated in reactions supplemented with topoisomerase II, as well as in reactions containing only topoisomerase II. In the presence of crude extracts of HeLa cells, VP-16, a specific inhibitor of topoisomerase II, blocked elongation of the nascent DNA during the replication of SV40 chromosomes. These results indicate that topoisomerase II plays a crucial role as a swivelase in the late stage of SV40 chromosome replication in vitro.     

A specific DNA unwinding activity associated with SV40 large T antigen

The incubation of highly purified large T antigen with relaxed, circular SV40 DNA in the presence of topoisomerase I (nicking closing enzyme) resulted in the introduction of negative superhelical turns in the DNA. ATP was not required for this reaction. A similar introduction of superhelical turns could also be obtained when a recombinant plasmid DNA (Y182), which contains sequences from both SV40 DNA and pBR322, was used. However, no effect was observed when relaxed pBR322 DNA, which does not contain SV40 DNA sequences, was incubated with T antigen in the presence of topoisomerase. These results are consistent with the hypothesis that large T antigen can recognize and unwind specific sequences on SV40 DNA.     

Class I major histocompatibility proteins as cell surface receptors for simian virus 40

Class I major histocompatibility complex proteins appear to be the major cell surface receptors for simian virus 40 (SV40), as implied by the following observations. Adsorption of SV40 to LLC-MK2 rhesus monkey kidney cells specifically inhibited binding of a monoclonal antibody (MAb) against class I human lymphocyte antigen (HLA) proteins. Conversely, pretreatment of LLC-MK2 cells with anti-HLA MAbs inhibited infection by SV40. The ability of anti-HLA to inhibit infection was greatly reduced when the order of addition of the anti-HLA and the virus was reversed. Infection was also inhibited by preincubating SV40 with purified soluble class I protein. Finally, human lymphoblastoid cells of the Daudi line, which do not express class I major histocompatibility complex proteins, were infected at relatively low levels with SV40 virions. In a control experiment, we found that pretreatment of cells with a MAb specific for the leukocytic-function-associated antigen LFA-3 actually enhanced infection. This finding may also support the premise that class I major histocompatibility complex proteins are receptors for SV40.     

Purification of DNA polymerase delta as an essential simian virus 40 DNA replication factor.

DNA replication from the SV40 origin can be reconstituted in vitro using purified SV40 large T antigen, cellular topoisomerases I and II, replication factor A (RF-A), proliferating cell nuclear antigen (PCNA), replication factor C (RF-C), and a phosphocellulose fraction (IIA) made from human cell extracts (S100). Fraction IIA contains all DNA polymerase activity required for replication in vitro in addition to other factors. A newly identified factor has been purified from fraction IIA. This factor is required for complete reconstitution of SV40 DNA replication and co-purifies with a PCNA-stimulated DNA polymerase activity. This DNA polymerase activity is sensitive to aphidicolin, but is not inhibited by butylanilinodeoxyadenosine triphosphate or by monoclonal antibodies which block synthesis by DNA polymerase alpha. The polymerase activity is synergistically stimulated by the combination of RF-A, PCNA, and RF-C in an ATP-dependent manner. Purified calf thymus polymerase delta can fully replace the purified factor in DNA replication assays. We conclude that this factor, required for reconstitution of SV40 DNA replication in vitro, corresponds to human DNA polymerase delta.     

Purification of a cellular replication factor, RF-C, that is required for coordinated synthesis of leading and lagging strands during simian virus 40 DNA replication in vitro.

Cell extracts (S100) derived from human 293 cells were separated into five fractions by phosphocellulose chromatography and monitored for their ability to support simian virus 40 (SV40) DNA replication in vitro in the presence of purified SV40 T antigen. Three fractions, designated I, IIA, and IIC, were essential. Fraction IIC contained the known replication factors topoisomerases I and II, but in addition contained a novel replication factor called RF-C. The RF-C activity, assayed in the presence of I, IIA, and excess amounts of purified topoisomerases, was detected in both cytosol and nuclear fractions, but was more abundant in the latter fraction. RF-C was purified from the 293 cell nuclear fraction to near homogeneity by conventional column chromatography. The reconstituted reaction mix containing purified RF-C could replicate SV40 origin-containing plasmid DNA more efficiently than could the S100 extract, and the products were predominantly completely replicated, monomer molecules. Interestingly, in the absence of RF-C, early replicative intermediates accumulated and subsequent elongation was aberrant. Hybridization studies with strand-specific, single-stranded M13-SV40 DNAs showed that in the absence of RF-C, abnormal DNA synthesis occurred preferentially on the lagging strand, and leading-strand replication was inefficient. These products closely resembled those previously observed for SV40 DNA replication in vitro in the absence of proliferating-cell nuclear antigen. These results suggest that an elongation complex containing RF-C and proliferating-cell nuclear antigen is assembled after formation of the first nascent strands at the replication origin. Subsequent synthesis of leading and lagging strands at a eucaryotic DNA replication fork can be distinguished by different requirements for multiple replication components, but we suggest that even though the two polymerases function asymmetrically, they normally progress coordinately.     

Identification of cellular proteins required for simian virus 40 DNA replication

Study of the proteins involved in DNA replication of a model system such as SV40 is a first step in understanding eukaryotic chromosomal replication. Using a cell-free system that is capable of replicating plasmid DNA molecules containing the SV40 origin of replication, we conducted a series of systematic fractionation-reconstitution experiments for the purpose of identifying and characterizing the cellular proteins involved in SV40 DNA replication. In addition to the one viral-encoded replication protein, T antigen, we have identified and begun to characterize at least six cellular components from a HeLa cytoplasmic extract that are absolutely required for SV40 DNA replication in vitro. These include: (i) two partially purified fractions, CF IC and CF IIA, and (ii) four proteins that have been purified to near homogeneity, replication protein-A, proliferating cell nuclear antigen, DNA polymerase alpha-primase complex, and topoisomerase (I and II). Replication protein-A is a multi-subunit protein that has single-stranded DNA binding activity and is required for a T antigen-dependent, origin-dependent unwinding reaction which may be an important early step in initiation of replication. Fraction CF IC can stimulate this unwinding reaction, suggesting that it also may function during initiation. Proliferating cell nuclear antigen, DNA polymerase alpha-primase, and CF IIA all appear to be involved in elongation of nascent chains.     

Patterns of strongly protein-associated simian virus 40 DNA replication intermediates resulting from exposures to specific topoisomerase poisons

Exposure of infected CV-1 cells to specific type I and type II topoisomerase poisons caused strong protein association with distinct subsets of simian virus 40 (SV40) DNA replication intermediates. On the basis of the known specificity and mechanisms of action of these drugs, the proteins involved are assumed to be the respective topoisomerases. Camptothecin, a topoisomerase I poison, caused strong protein association with form II (relaxed circular) and form III (linear) viral genomes and replication intermediates having broken DNA replication forks but not with form I (superhelical) viral DNA or normal late replication intermediates which were present. In contrast, type II topoisomerase poisons caused completely replicated forms and late viral replication forms to be tightly bound to protein--some to a greater extent than others. Different type II topoisomerase inhibitors caused distinctive patterns of protein association with the replication intermediates present. Both intercalating and nonintercalating type II topoisomerase poisons caused a small amount of form I (superhelical) SV40 DNA to be protein-associated in vivo. The protein complex with form I viral DNA was entirely drug-dependent and strong, but apparently noncovalent. The protein associated with form I DNA may represent a drug-stabilized "topological complex" between type II topoisomerase and SV40 DNA.     

Immunoprecipitation of some forms of simian virus 40 large-T antigen by antibodies to synthetic peptides.

Antibodies were raised against six synthetic peptides corresponding to overlapping amino acid sequences (106 through 145) from a putative DNA binding domain in simian virus 40 (SV40) large-T antigens. All six antipeptide sera immunoprecipitated large-T from crude extracts of SV40-transformed cells, but the efficiency varied widely; in general, antibodies to the longer peptides produced the strongest anti-large-T activity. Antisera were purified by immunoaffinity chromatography on immobilized peptide. The purified antisera recognized only some forms of large-T; full-sized large-T from transformed cells, super-T from SV3T3 C120 cells, and 70,000-dalton T-antigen from Taq-BamHI cells were immunoprecipitated, whereas large-T from productively infected cells reacted irreproducibly, and the full-sized protein, synthesized in vitro or eluted from sodium dodecyl sulfate-containing gels, and the 33,000- and 22,000-dalton truncated large-Ts from Swiss SV3T3 and MES2006 cells, respectively, were not immunoprecipitated. This pattern of reactivity was explained when extracts were fractionated by sucrose density centrifugation, and it was found that only rapidly sedimenting forms of large-T were immunoprecipitated by the antipeptide sera; that is, large-T complexed with nonviral T antigen was detected, whereas lighter forms were not detected. Cascade immunoprecipitations did not support the view that this result was caused by the low affinity of the peptide antisera for large-T, and Western blotting experiments confirmed that the peptide antisera react directly with immobilized, monomeric large-T but not with nonviral T antigen. Immunoprecipitation assays to detect large-T:nonviral T antigen complexes bound specifically to fragments of SV40 DNA showed that under conditions of apparent antibody excess, DNA still bound to the complex.     

Interaction between the N-terminus of human topoisomerase I and SV40 large T antigen.

We have attempted to identify human topoisomerase I-binding proteins in order to gain information regarding the cellular roles of this protein and the cytotoxic mechanisms of the anticancer drug camptothecin, which specifically targets topoisomerase I. In the course of this work we identified an interaction between the N-terminus of human topoisomerase I and the SV40 T antigen that is detectable in vitro using both affinity chromatography and co-immunoprecipitation. Additional results indicate that this interaction does not require intermediary DNA or stoichiometric quantities of other proteins. Furthermore, the interaction is detectable in vivo using a yeast two-hybrid assay. Two binding sites for T antigen are apparent on the topoisomerase I protein: one consisting of amino acids 1-139, the other present in the 383-765 region of the protein. Interestingly, nucleolin, which binds the 166-210 region of topoisomerase I, is able to bind an N-terminal fragment of topoisomerase I concurrently with T antigen. Taken together with our prior identification of nucleolin as a topoisomerase I-binding protein, the current results suggest that helicase-binding is a major role of the N-terminus of human topoisomerase I and that the resultant helicase-topoisomerase complex may function as a eukaryotic gyrase.     

Biochemical activities of T-antigen proteins encoded by simian virus 40 A gene deletion mutants.

We have analyzed T antigens produced by a set of simian virus 40 (SV40) A gene deletion mutants for ATPase activity and for binding to the SV40 origin of DNA replication. Virus stocks of nonviable SV40 A gene deletion mutants were established in SV40-transformed monkey COS cells. Mutant T antigens were produced in mutant virus-infected CV1 cells. The structures of the mutant T antigens were characterized by immunoprecipitation with monoclonal antibodies directed against distinct regions of the T-antigen molecule. T antigens in crude extracts prepared from cells infected with 10 different mutants were immobilized on polyacrylamide beads with monoclonal antibodies, quantified by Coomassie blue staining, and then assayed directly for T antigen-specific ATPase activity and for binding to the SV40 origin of DNA replication. Our results indicate that the T antigen coding sequences required for origin binding map between 0.54 and 0.35 map units on the SV40 genome. In contrast, sequences closer to the C terminus of T antigen (between 0.24 and 0.20 map units) are required for ATPase activity. The presence of the ATPase activity correlated closely with the ability of the mutant viruses to replicate and to transform nonpermissive cells. The origin binding activity was retained, however, by three mutants that lacked these two functions, indicating that this activity is not sufficient to support either cellular transformation or viral replication. Neither the ATPase activity nor the origin binding activity correlated with the ability of the mutant DNA to activate silent rRNA genes or host cell DNA synthesis.     

Simian virus 40 DNA replication in vitro: purification and characterization of replication factors from mouse cells.

We have previously developed simian virus 40 (SV40) DNA replication system in vitro (Ariga and Sugano, J. Virol. 48, 481, 1983). This system is composed of human HeLa or mouse FM3A nuclear extract and cytoplasmic extract of SV40 infected CosI cells. Here FM3A nuclear extract was fractionated by DEAE Sephacel and single-stranded DNA cellulose chromatography into three components required for accurate in vitro SV40 DNA replication. One fraction (A fraction) contained DNA polymerase-primase, and the second component (B fraction) contained DNA topoisomerase. Third component was further purified to near homogenuity using DEAE-Sephacel, single-stranded DNA cellulose, and glycerol gradient centrifugation. The purified protein (named factor I) bound to the origin containing fragment of SV40 DNA. The factor I enhanced the initiation of SV40 DNA replication catalyzed by SV40 infected CosI cytoplasm alone. When all four fractions consisting of A, B fractions, factor I, and SV40 infected CosI cytoplasm were mixed together, the system was reconstituted, meaning that initiation and subsequent elongation were completed to generate the full sized daughter molecules.     

Site-dependent inhibition by single O6-methylguanine bases of SV40 T-antigen interactions with the viral origin of replication.

The effects of O6-methylguanine on the reactions involved in initiation of DNA replication were investigated by measuring the interactions of SV40 T antigen with oligonucleotides substituted with the methylated base. O6-Methylguanine residues were positioned in either binding site I or binding site II of the SV40 origin of replication. Binding of purified T antigen, measured by both nitrocellulose filter binding and delayed oligonucleotide migration, was unaffected by the presence of seven methylated bases in binding site II. Single substitutions within binding site I were sufficient to inhibit T-antigen binding, and the extent of inhibition was dependent on the position of O6-methylguanine in the DNA sequence. Unwinding by T antigen was analyzed by measuring displacement of a single-stranded oligonucleotide from similarly substituted, partially duplex substrates. The presence of three O6-methylguanine residues in binding site I facilitated the helicase activity of T antigen. In contrast, single O6-methylguanine bases inhibited unwinding. A correlation was observed between the position of the methylated base and the inhibition of both binding and unwinding by T antigen.