DNA-binding properties of simian virus 40 T-antigen mutants defective in viral DNA replication.

Three simian virus 40 (SV40)-transformed monkey cell lines, C2, C6, and C11, producing T-antigen variants that are unable to initiate viral DNA replication, were analyzed with respect to their affinity for regulatory sequences at the viral origin of replication. C2 and C11 T antigens both bound specifically to sequences at sites 1 and 2 at the viral origin region, whereas C6 T antigen showed no specific affinity for any viral DNA sequences under all conditions tested. Viral DNA sequences encoding the C6 T antigen have recently been cloned out of C6 cells and used to transform an established rat cell line. T antigen from several cloned C6-SV40-transformed rat lines failed to bind specifically to the origin. C6 DNA contains three mutations: two located close to the amino terminus of T antigen at amino acid positions 30 and 51 and a third located internally at amino acid position 153. Two recombinant SV40 DNA mutants were prepared containing either the amino-terminal mutations at positions 30 and 51 (C6-1) or the internally located mutation at position 153 (C6-2) and used to transform Rat 2 cells. Whereas T antigen from C6-2-transformed cells lacked any specific affinity for these sequences. Therefore, the single mutation at amino acid position 153 (Asn leads to Thr) is sufficient to abolish the origin-binding property of T antigen. A T antigen-specific monoclonal antibody, PAb 100, which had been previously shown to immunoprecipitate an immunologically distinct origin-binding subclass of T antigen, recognized wild-type or C6-1 antigens, but failed to react with C6 or C6-2 T antigens. These results indicate that viral replication function comprises properties of T antigen that exist in addition to its ability to bind specifically to the SV40 regulatory sequences. Furthermore, it is concluded from these data that specific viral origin binding is not a necessary feature of the transforming function of T antigen.     

Large T-antigen double hexamers imaged at the simian virus 40 origin of replication

The initial step of simian virus 40 (SV40) DNA replication is the binding of the large tumor antigen (T-Ag) to the SV40 core origin. In the presence of Mg(2+) and ATP, T-Ag forms a double-hexamer complex covering the complete core origin. By using electron microscopy and negative staining, we visualized for the first time T-Ag double hexamers bound to the SV40 origin. Image processing of side views of these nucleoprotein complexes revealed bilobed particles 24 nm long and 8 to 12 nm wide, which indicates that the two T-Ag hexamers are oriented head to head. Taking into account all of the biochemical data known on the T-Ag-DNA interactions at the replication origin, we present a model in which the DNA passes through the inner channel of both hexamers. In addition, we describe a previously undetected structural domain of the T-Ag hexamer and thereby amend the previously published dimensions of the T-Ag hexamer. This domain we have determined to be the DNA-binding domain of T-Ag.     

Mutational analysis of simian virus 40 T-antigen primosome activities in viral DNA replication

The recruitment of DNA polymerase alpha-primase (pol-prim) is a crucial step in the establishment of a functional replication complex in eukaryotic cells, but the mechanism of pol-prim loading and the composition of the eukaryotic primosome are poorly understood. In the model system for simian virus 40 (SV40) DNA replication in vitro, synthesis of RNA primers at the origin of replication requires only the viral tumor (T) antigen, replication protein A (RPA), pol-prim, and topoisomerase I. On RPA-coated single-stranded DNA (ssDNA), T antigen alone mediates priming by pol-prim, constituting a relatively simple primosome. T-antigen activities proposed to participate in its primosome function include DNA helicase and protein-protein interactions with RPA and pol-prim. To test the role of these activities of T antigen in mediating priming by pol-prim, three replication-defective T antigens with mutations in the ATPase or helicase domain have been characterized. All three mutant proteins interacted physically and functionally with RPA and pol-prim and bound ssDNA, and two of them displayed some helicase activity. However, only one of these, 5030, mediated primer synthesis and elongation by pol-prim on RPA-coated ssDNA. The results suggest that a novel activity, present in 5030 T antigen and absent in the other two mutants, is required for T-antigen primosome function.     

Role of the hydrophilic channels of simian virus 40 T-antigen helicase in DNA replication

The simian virus 40 (SV40) hexameric helicase consists of a central channel and six hydrophilic channels located between adjacent large tier domains within each hexamer. To study the function of the hydrophilic channels in SV40 DNA replication, a series of single-point substitutions were introduced at sites not directly involved in protein-protein contacts. The mutants were characterized biochemically in various ways. All mutants oligomerized normally in the absence of DNA. Interestingly, 8 of the 10 mutants failed to unwind an origin-containing DNA fragment and nine of them were totally unable to support SV40 DNA replication in vitro. The mutants fell into four classes based on their biochemical properties. Class A mutants bound DNA normally and had normal ATPase and helicase activities but failed to unwind origin DNA and support SV40 DNA replication. Class B mutants were compromised in single-stranded DNA and origin DNA binding at low protein concentrations. They were defective in helicase activity and unwinding of the origin and in supporting DNA replication. Class C and D mutants possessed higher-than-normal single-stranded DNA binding activity at low protein concentrations. The class C mutants failed to separate origin DNA and support DNA replication. The class D mutants unwound origin DNA normally but were compromised in their ability to support DNA replication. Taken together, these results suggest that the hydrophilic channels have an active role in the unwinding of SV40 DNA from the origin and the placement of the resulting single strands within the helicase.     

DNA replication and chromatin structure of simian virus 40 insertion mutants.

Insertion of DNA segments into the nuclease-sensitive region of simian virus 40 alters both replication efficiency and chromatin structure. Mutants containing large insertions between the simian virus 40 origin of replication (ori site) and the 21-base-pair repeated sequences replicated poorly when assayed by transfection into COS-1 cells. Replication of mutants with shorter insertions was moderately reduced. This effect was cis-acting and independent of the nucleotide sequence of the insert. The nuclease-sensitive chromatin structure was retained in these mutants, but the pattern of cleavage sites was displaced in the late direction from the ori site. New cleavage sites appeared within the inserted sequences, suggesting that information specifying the nuclease-sensitive chromatin structure is located on the late side of the inserts. Accessibility to BglI (which cleaves within the ori site) was reduced in the larger insertion mutants. These results support the conclusion that efficient function of the viral origin of replication is correlated with its proximity to an altered chromatin structure.     

In vitro initiation of DNA replication in simian virus 40 chromosomes

A soluble system has been developed that can initiate DNA replication de novo in simian virus 40 (SV40) chromatin isolated from virus-infected monkey cells as well as in circular plasmid DNA containing a functional SV40 origin of replication (ori). Initiation of DNA replication in SV40 chromatin required the soluble fraction from a high-salt nuclear extract of SV40-infected cells, a low-salt cytosol fraction, polyethylene glycol, and a buffered salts solution containing all four standard deoxyribonucleoside triphosphates. Purified SV40 large tumor antigen (T-ag) partially substituted for the high-salt nucleosol, and monoclonal antibodies directed against SV40 T-ag inhibited DNA replication. Replication began at ori and proceeded bidirectionally to generate replicating DNA intermediates in which the parental strands remained covalently closed, as observed in vivo. Partial inhibition of DNA synthesis by aphidicolin resulted in accumulation of newly initiated replicating intermediates in this system, a phenomenon not observed under conditions that supported completion of replication only. However, conditions that were optimal for initiation of replication repressed conversion of late-replicating intermediates into circular DNA monomers. Most surprising was the observation that p-n-butylphenyl-dGTP, a potent and specific inhibitor of DNA polymerase-alpha, failed to inhibit replication of SV40 chromatin under conditions that completely inhibited replication of plasmid DNA containing the SV40 ori and either purified or endogenous DNA polymerase-alpha activity. In contrast, all of these DNA synthesis activities were inhibited equally by aphidicolin. Therefore, DNA replication in mammalian cells is carried out either by DNA polymerase-alpha that bears a unique association with chromatin or by a different enzyme such as DNA polymerase-delta.     

Dimerization of simian virus 40 T-antigen hexamers activates T-antigen DNA helicase activity.

Chromosomal DNA replication in higher eukaryotes takes place in DNA synthesis factories containing numerous replication forks. We explored the role of replication fork aggregation in vitro, using as a model the simian virus 40 (SV40) large tumor antigen (T antigen), essential for its DNA helicase and origin-binding activities. Previous studies have shown that T antigen binds model DNA replication forks primarily as a hexamer (TAgH) and to a lesser extent as a double hexamer (TAgDH). We find that DNA unwinding in the presence of ATP or other nucleotides strongly correlates with the formation of TAgDH-DNA fork complexes. TAgH- and TAgDH-fork complexes were isolated, and the TAgDH-bound fork was denatured at a 15-fold-higher rate during the initial times of unwinding. TAgDH bound preferentially to a DNA substrate containing a 50-nucleotide bubble, indicating the bridging of each single-stranded DNA/duplex DNA junction, and this DNA molecule was also unwound at a high rate. Both the TAgH- and TAgDH-fork complexes were relatively stable, with the half-life of the TAgDH-fork complex greater than 40 min. Our data therefore indicate that the linking of two viral replication forks serves to activate DNA replication.     

Inhibition of simian virus 40 DNA replication by specific modification of T-antigen with oxidized ATP

We covalently bound periodate-oxidized ATP (oATP) to purified simian virus 40 (SV40) large T-antigen and determined the effect of this modification on viral DNA replication and three other biochemical activities of T-antigen. The oATP bound specifically to T-antigen, inhibiting the ATPase activity and preventing T-antigen from activating SV40 DNA replication in vitro. In contrast, binding of oATP had no effect on the DNA-binding activity of T-antigen nor on its ability to form a complex with DNA polymerase alpha. These results provide direct biochemical evidence suggesting that the T-antigen ATPase activity is necessary for viral DNA replication.     

Human topoisomerase I promotes initiation of simian virus 40 DNA replication in vitro

Addition of purified human topoisomerase I (topo I) to simian virus 40 T antigen-driven in vitro DNA replication reactions performed with topo I-deficient extracts results in a greater than 10-fold stimulation of completed molecules as well as a more than 3-fold enhancement of overall DNA replication. To further characterize this stimulation, we first demonstrate that bovine topo I but not Escherichia coli topo I can also enhance DNA replication. By using several human topo I mutants, we show that a catalytically active form of topo I is required. To delineate whether topo I influences the initiation or the elongation step of replication, we performed delayed pulse, pulse-chase, and delayed pulse-chase experiments. The results illustrate that topo I cannot promote the completion of partially replicated molecules but is needed from the beginning of the reaction to initiate replication. Competitive inhibition experiments with the topo I binding T antigen fragment 1-246T and a catalytically inactive topo I mutant suggest that part of topo I's stimulation of replication is mediated through a direct interaction with T antigen. Collectively, our data indicate that topo I enhances the synthesis of fully replicated DNA molecules by forming essential interactions with T antigen and stimulating initiation.     

Structural topography of simian virus 40 DNA replication.

Applying an in situ cell fractionation procedure, we analyzed structural systems of the cell nucleus for the presence of mature and replicating simian virus 40 (SV40) DNA. Replicating SV40 DNA intermediates were tightly and quantitatively associated with the nuclear matrix, indicating that elongation processes of SV40 DNA replication proceed at this structure. Isolated nuclei as well as nuclear matrices were able to continue SV40 DNA elongation under replication conditions in situ, arguing for a coordinated and functional association of SV40 DNA and large T molecules at nuclear structures. SV40 DNA replication also was terminated at the nuclear matrix. While the bulk of newly synthesized, mature SV40 DNA molecules then remained at this structure, some left the nuclear matrix and accumulated at the chromatin.     

Non-specific termination of simian virus 40 DNA replication.

Axial X-ray diffraction patterns have been studied from relaxed, contracted and rigor vertebrate striated muscles at different sarcomere lengths to determine which features of the patterns depend on the interaction of actin and myosin. The intensity of the myosin layer lines in a live, relaxed muscle is sometimes less in a stretched muscle than in the muscle at rest-length; the intensity depends not only on the sarcomere length but on the time that has elapsed since dissection of the muscle. The movement of cross-bridges giving rise to these intensity changes are not caused solely by the withdrawal of actin from the A-band.When a muscle contracts or passes into rigor many changes occur that are independent of the sarcomere length: the myosin layer lines decrease in intensity to about 30% of their initial value when the muscle contracts, and disappear completely when the muscle passes into rigor. Both in contracting and rigor muscles at all sarcomere lengths the spacings of the meridional reflections at 143 Å and 72 Å are 1% greater than from a live relaxed muscle at rest-length. It is deduced that the initial movement of cross-bridges from their positions in resting muscle does not depend on the interaction of each cross-bridge with actin, but on a conformational change in the backbone of the myosin filament: occurring as a result of activation. The possibility is discussed that the conformational change occurs because the myosin filament, like the actin filament, has an activation control mechanism. Finally, all the X-ray diffraction patterns are interpreted on a model in which the myosin filament can exist in one of two possible states: a relaxed state which gives a diffraction pattern with strong myosin layer lines and an axial spacing of 143.4 Å, and an activated state which gives no layer lines but a meridional spacing of 144.8 Å.     

Initiation of simian virus 40 DNA replication in vitro.

Exogenously added simian virus 40 (SV40) DNA can be replicated semiconservatively in vitro by a mixture of a soluble extract of HeLa cell nuclei and the cytoplasm from SV40-infected CosI cells. When cloned DNA was used as a template, the clone containing the SV40 origin of DNA replication was active, but a clone lacking the SV40 origin was inactive. The major products of the in vitro reaction were form I and form II SV40 DNAs and a small amount of form III. DNA synthesis in extracts began at or near the in vivo origin of SV40 DNA synthesis and proceeded bidirectionally. The reaction was inhibited by the addition of anti-large T hamster serum, aphidicolin, or RNase but not by ddNTP. Furthermore, this system was partially reconstituted between HeLa nuclear extract and the semipurified SV40 T antigen instead of the CosI cytoplasm. It is clear from these two systems that the proteins containing SV40 T antigen change the nonspecific repair reaction performed by HeLa nuclear extract alone to the specific semiconservative DNA replication reaction. These results show that these in vitro systems closely resemble SV40 DNA replication in vivo and provide an assay that should be useful for the purification and subsequent characterization of viral and cellular proteins involved in DNA replication.     

Regulatory mutants of simian virus 40: constructed mutants with base substitutions at the origin of DNA replication.

Mutants of simian virus 40 (SV40) with base substitutions at or near the origin of replication of the viral genome have been constructed by bisulfite mutagenesis at the BglI restriction site of SV40 DNA, followed by transfection of cells with the BglI-resistant (BglI^r) DNA so generated. Based on plaque morphology at different temperatures, the resulting BglI^r mutants could be classified into four-groups. Class I mutants (designated ar for “altered restriction”) were indistinguishable from wild-type SV40; class II mutants (designated shp for “sharp plaque”) produced small, sharp-edged plaques; class III mutants (designated sp for “small plaque”) produced small plaques at 32 °C, 37 °C and 40 °C; and class IV mutants (designated cs for “cold sensitive”) produced small plaques at 32 °C and wild-type plaques at 37 °C and 40 °C. That the altered plaque morphology of sp and cs mutants was related to mutation at the BglI restriction site was demonstrated by co-reversion to wild-type of the plaque phenotype and BglI sensitivity. The nucleotide sequence around the original BglI site was determined in the DNA from one mutant of each class. In each case a different base-pair substitution was found, at a site outside sequences coding for SV40 proteins. When rates of replication of mutant DNAs were measured during productive infection, ar mutant DNA was synthesized at a rate comparable to that of wild-type SV40 DNA, shp mutant DNA was made at a rate exceeding that of wild-type, sp mutant DNA was synthesized at a lower rate than that of wild type. and cs mutant DNA synthesis was reduced at 32 °C, but about the same as the wild-type rate at 40 °C. These patterns of mutant DNA synthesis were unaltered in cells co-infected with mutant and wild-type virus, i.e. the defects in DNA synthesis were not trans-complementable. We conclude that the defective mutants have single base-pair changes in a cis element that determines the rate of viral DNA replication, presumably within the origin signal itself.     

Phosphorylation of T-antigen and control T-antigen expression in cells transformed by wild-type and tsA mutants of simian virus 40.

Chinese hamster lung (CHL) cells transformed by wild-type simian virus 40 (cell line CHLWT15) or transformed by the simian virus 40 mutants tsA30 (cell lines CHLA30L1 and CHLA30L2) or tsA239 (cell line CHLA239L1) were used to determine the rates of turnover and synthesis of the T-antigen protein and the rate of turnover of the phosphate group(s) attached to the T-antigen at both the permissive and restrictive temperatures. The phosphate group turned over several times within the lifetime of the protein to which it was attached, with the exception of the phosphate group in the tsA transformants at 40 degrees C, which turned over at the same rate as the T-antigen protein. The steady-state levels of the T-antigens (molecular weights, 92,000 [92K] and 17K) and the amount of simian virus 40-specific RNA was also determined in each of the lines. The CHLA30L1 line contained two to three times more early simian virus 40 RNA than the CHLA30L2 line; although neither line formed colonies in agar at 40 degrees C, CHLA30L1 overgrew a normal monolayer at 40 degrees C. The rate of 92K-T-antigen synthesis was 1.5 times faster in CHLA30L1 than in CHLA30L2 at 33 degrees C and 4 times faster at 40 degrees C. The different phenotype of these two presumably isogenic cell lines seem to be related to the levels of the T-antigens. The ratios of the 92K T-antigen to the 17K T-antigens were similar in the two lines. Transformed CHL cell lines, unlike transformed mouse 3T3 cell lines, were found to contain very small amounts of the 56K T-antigen.     

Cooperative assembly of simian virus 40 T-antigen hexamers on functional halves of the replication origin.

The cofactor ATP stimulates the formation of T-antigen double hexamers on the simian virus 40 core origin of replication (I. A. Mastrangelo, P. V. C. Hough, J. S. Wall, M. Dodson, F. B. Dean, and J. Horwitz, Nature [London] 338:658-662, 1989). We report here the pathway for the assembly of hexamers and double hexamers on the core origin. ATP triggers the cooperative assembly of hexamers on the early and late halves of the origin even when they are completely isolated. Hexamer assembly nucleates at T-antigen recognition pentanucleotides in the early half of the origin. In intact origins, assembly of the first hexamer on the early half of the origin cooperatively stimulates the assembly of a second hexamer on the adjacent late half of the origin. Thus, monomer-monomer and hexamer-hexamer interactions of T antigen, allosterically activated by ATP, constitute two distinct types of cooperative interaction with the origin. Finally, we show that the assembly of T-antigen hexamers on isolated half origins leads to the same array of structural changes that T antigen induces in intact origins. We conclude that the origin is divided into complementary halves that each promote the assembly of functional T-antigen hexamers.     

Allosteric control of simian virus 40 T-antigen binding to viral origin DNA.

Simian virus 40 (SV40) large tumor antigen (T antigen) possesses several biochemical activities localized in different domains of the protein. These activities include sequence-specific binding to two major sites, I and II, in the SV40 control region, ATPase, and nucleotide-binding activity. In the present communication, we present evidence that specific binding of immunopurified T antigen to SV40 DNA is markedly inhibited by low concentrations of ATP, dATP, GTP, and dGTP. The inhibition is reversible after removal of the nucleotide, suggesting that simple nucleotide binding rather than a covalent modification of T antigen in the presence of ATP is responsible for the inhibition. The results suggest that T antigen may assume two conformations, one active and one inactive in binding to the SV40 origin of replication. In the presence of purine nucleoside triphosphates, the inactive conformation is favored.     

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: identification of multiple stages of initiation.

A cell-free DNA replication system dependent upon five purified cellular proteins, one crude cellular fraction, and the simian virus 40 (SV40)-encoded large tumor antigen (T antigen) initiated and completed replication of plasmids containing the SV40 origin sequence. DNA synthesis initiated at or near the origin sequence after a time lag of approximately 10 min and then proceeded bidirectionally from the origin to yield covalently closed, monomer daughter molecules. The time lag could be completely eliminated by a preincubation of SV40 ori DNA in the presence of T antigen, a eucaryotic single-stranded DNA-binding protein (replication factor A [RF-A]), and topoisomerases I and II. In contrast, if T antigen and the template DNA were incubated alone, the time lag was only partially decreased. Kinetic analyses of origin recognition by T antigen, origin unwinding, and DNA synthesis suggest that the time lag in replication was due to the formation of a complex between T antigen and DNA called the T complex, followed by formation of a second complex called the unwound complex. Formation of the unwound complex required RF-A. When origin unwinding was coupled to DNA replication by the addition of a partially purified cellular fraction (IIA), DNA synthesis initiated at the ori sequence, but the template DNA was not completely replicated. Complete DNA replication in this system required the proliferating-cell nuclear antigen and another cellular replication factor, RF-C, during the elongation stage. In a less fractionated system, another cellular fraction, SSI, was previously shown to be necessary for reconstitution of DNA replication. The SSI fraction was required in the less purified system to antagonize the inhibitory action of another cellular protein(s). This inhibitor specifically blocked the earliest stage of DNA replication, but not the later stages. The implications of these results for the mechanisms of initiation and elongation of DNA replication are discussed.     

Primer-DNA formation during simian virus 40 DNA replication in vitro.

Studies of simian virus 40 (SV40) DNA replication in vitro have identified a small (approximately 30-nucleotide) RNA-DNA hybrid species termed primer-DNA. Initial experiments indicated that T antigen and the polymerase alpha-primase complex are required to form primer-DNA. Proliferating cell nuclear antigen, and presumably proliferating cell nuclear antigen-dependent polymerases, is not needed to form this species. Herein, we present an investigation of the stages at which primer-DNA functions during SV40 DNA replication in vitro. Hybridization studies indicate that primer-DNA is initially formed in the origin region and is subsequently synthesized in regions distal to the origin. At all time points, primer-DNA is synthesized from templates for lagging-strand DNA replication. These studies indicate that primer-DNA functions during both initiation and elongation stages of SV40 DNA synthesis. Results of additional experiments suggesting a precursor-product relationship between formation of primer-DNA and Okazaki fragments are presented.