Nucleoprotein Complexes Containing Replicating Simian Virus 40 DNA: Comparison with Polyoma Nucleoprotein Complexes

Procedures for isolating nucleoprotein complexes containing replicating polyoma DNA from infected mouse cells were used to prepare short-lived nucleoprotein complexes (r-SV40 complexes) containing replicating simian virus 40 (SV40) DNA from infected monkey cells. Like the polyoma complexes, r-SV40 complexes were only partially released from nuclei by cell lysis but could be extracted from nuclei by prolonged treatment with solutions containing Triton X-100. r-SV40 complexes sedimented faster than complexes containing SV40 supercoiled DNA (SV40 complex) in sucrose gradients, and both types of SV40 nucleoprotein complexes sedimented ahead of polyoma complexes containing supercoiled polyoma DNA (py complex). The sedimentation rates of py complex and SV40 complex were 56 and 61S, respectively, based on the sedimentation rate of the mouse large ribosomal subunit as a marker. r-SV40 complexes sedimented as multiple peaks between 56 and 75S. Sedimentation and buoyant density measurements indicated that protein is bound to all forms of SV40 DNA at about the same ratio of protein to DNA (1-2/1) as was reported for polyoma nucleoproteins.     

Association of Simian Virus 40 T Antigen with Simian Virus 40 Nucleoprotein Complexes

Viral nucleoprotein complexes were extracted from the nuclei of simian virus 40 (SV40)-infected TC7 cells by low-salt treatment in the absence of detergent, followed by sedimentation on neutral sucrose gradients. Two forms of SV40 nucleoprotein complexes, those containing SV40 replicative intermediate DNA and those containing SV40 (I) DNA, were separated from one another and were found to have sedimentation values of 125 and 93S, respectively. [(35)S]methioninelabeled proteins in the nucleoprotein complexes were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. In addition to VP1, VP3, and histones, a protein with a molecular weight of 100,000 (100K) is present in the nucleoprotein complexes containing SV40 (I) DNA. The 100K protein was confirmed as SV40 100K T antigen, both by immunoprecipitation with SV40 anti-T serum and by tryptic peptide mapping. The 100K T antigen is predominantly associated with the SV40 (I) DNA-containing complexes. The 17K T antigen, however, is not associated with the SV40 (I) DNA-containing nucleoprotein complexes. The functional significance of the SV40 100K T antigen in the SV40 (I) DNA-containing nucleoprotein complexes was examined by immunoprecipitation of complexes from tsA58-infected TC7 cells. The 100K T antigen is present in nucleoprotein complexes extracted from cells grown at the permissive temperature but is clearly absent from complexes extracted from cells grown at the permissive temperature and shifted up to the nonpermissive temperature for 1 h before extraction, suggesting that the association of the 100K T antigen with the SV40 nucleoprotein complexes is involved in the initiation of SV40 DNA synthesis.     

Intracellular forms of simian virus 40 nucleoprotein complexes. IV. Micrococcal nuclease digestion.

The structures of DNAs present in various intracellular forms of simian virus 40 (SV40) nucleoprotein complexes were analyzed by micrococcal nuclease digestion. The results showed that the 70S SV40 chromatin was completely sensitive to nuclease digestion, whereas CsCl gradient-purified mature virion was completely resistant. Virion assembly intermediates with different degrees of virion maturation showed intermediate resistance, and three products were found: nucleosomal DNA fragments, representing the fraction of intermediates that were sensitive to nuclease; linear SV40 genome-sized DNA, representing the more mature intermediates that contained one or limited defects in the capsid shell; and supercoiled SV40, which was derived from mature virions. These digestion products, however, remained associated with capsid shells after nuclease digestion. These results were consistent with the model in which maturation of the SV40 virion is achieved through the organization of capsid proteins that accumulate around SV40 chromatin. Mild digestion of SV40 nucleoprotein complexes with micrococcal nuclease revealed the difference in nucleosome repeat length between SV40 chromatin and virion assembly intermediates. A novel DNA fragment of about 75 nucleotides was observed early in nuclease digestion.     

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.     

Salt-resistant association of simian virus 40 T antigen with simian virus 40 DNA in nucleoprotein complexes.

Treatment of nucleoprotein complexes (NPCs) from simian virus 40 (SV40)-infected TC7 cells with NaCl (1 or 2 M) or guanidine-hydrochloride (1 or 2 M) resulted in a significant fraction of T antigen still associated with SV40 (I) DNA. Immunoprecipitation of the salt-treated NPCs with SV40 anti-T serum indicated that T antigen is preferentially associated with SV40 (I) DNA rather than with SV40 (II) DNA. Treatment of the NPCs with 4 M guanidine-hydrochloride, however, resulted in a substantial decrease in the amount of SV40 (I) and (II) DNA associated with T antigen. As the temperature was increased to 37 degrees C during incubation of NPCs with NaCl or guanidine-hydrochloride, there was a decrease in the amount of SV40 (I) and (II) DNA immunoprecipitated with SV40 anti-T serum. In the absence of salt, temperature had no effect on the association of T antigen with the SV40 DNA in the NPCs. Treatment of NPCs from SV40 wildtype or tsA58-infected cells grown at the permissive temperature with 1 or 2 M NaCl indicated that tsA T antigen has the same sensitivities as wild-type T antigen to high salt treatment when bound to DNA in NPCs. Characterization of the proteins associated with SV40 (I) DNA after high salt treatment revealed that, in addition to T antigen, a certain amount of viral capsid proteins VP1 and VP3 remained associated with the DNA. Complexes containing SV40 (I) DNA had a sedimentation value of 53S after 1 M NaCl treatment and 43S after 2 M NaCl treatment.     

Chromatin-like structures in polyoma virus and simian virus 10 lytic cycle.

Nucleoprotein complexes containing viral DNA and cellular histones were extracted from nuclei of permissive cells infected with polyoma virus or simian virus 40 (SV40) and examined by electron microscopy. Polyoma and SV40 nucleoprotein complexes are almost identical. They appear as relaxed circular molecules consisting of 20 to 21 globular particles interconnected by thin filaments. Their contour length in 0.02 M salt is 2.7 times shorter than that of viral DNA form I obtained after dissociation of the proteins in 1 M NaCl. The nucleosomes have an average diameter of 12.5 nm. Each nucleosome contains 175 to 205 DNA base pairs condensed fivefold in length. The nucleosomes are regularly spaced on the circular molecule. The internucleosomal filaments are made of naked DNA, and each filament contains about 55 base pairs. The partial sensitivity of the nucleoprotein complex to cleavage by EcoR1 endonuclease suggests that the nucleosomes are not formed at specific sites on the viral genome. Faster sedimenting nucleoprotein complexes containing replicative intermediates were studied. Isopycnic centrifugation in metrizamide gradients in the absence of aldehyde fixation showed that these molecules conserved the same DNA-to-protein ratio as the form I DNA-containing complexes.     

Replication of SV40 chromatin in extracts from eggs of Xenopus laevis.

Simian virus 40 (SV40) nucleoprotein complexes were prepared from lytically infected cells and used as primer-templates for DNA replication in protein extracts from Xenopus eggs. We found that nucleoprotein containing replicating SV40 DNA served as primer-template while nucleoprotein with nonreplicating SV40 DNA was ineffective. In vitro DNA synthesis begins with short DNA fragments ("Okazaki fragments") which are, in later steps, joined to give unit length SV40 DNA strands, suggesting that in vivo initiated rounds of replication are completed in vitro in the Xenopus system. This conclusion is supported by a restriction enzyme analysis showing that in vitro DNA synthesis occurs in fragments distal to the SV40 origin of replication. Our studies indicate that SV40 DNA replication in Xenopus extracts can be used an an experimental system to study the biochemistry of replicative DNA chain elongation in vitro.     

DNA polymerase alpha is associated with replicating SV40 nucleoprotein complexes.

Simian virus 40 (SV40) nucleoprotein complexes were extracted from nuclei of infected monkey cells and fractionated on neutral sucrose density gradients. Complexes which contained replicating SV40 DNA (95S) separated well from those containing closed circular supercoiled viral DNA (75S). DNA polymerase activity was associated with the replicating nucleoprotein complexes but not with the slower sedimenting complexes. This DNA polymerase activity coprecipitated with the nucleoprotein complexes in the presence of MgCl2 and remained associated with the 95S complexes. This DNA polymerase activity has been identified as primarily DNA polymerase alpha on the basis of its sedimentation behavior, optimum salt concentration, and sensitivity to N-ethylmaleimide. DNA polymerase gamma activity was also detected in the complexes, but DNA polymerase beta was not associated with the complexes.     

Electron microscopic single particle analysis of a tetrameric RuvA/RuvB/Holliday junction DNA complex

During the late stage of homologous recombination in prokaryotes, RuvA binds to the Holliday junction intermediate and executes branch migration in association with RuvB. The RuvA subunits form two distinct complexes with the Holliday junction: complex I with the single RuvA tetramer on one side of the four way junction DNA, and complex II with two tetramers on both sides. To investigate the functional roles of complexes I and II, we mutated two residues of RuvA (L125D and E126K) to prevent octamer formation. An electron microscopic analysis indicated that the mutant RuvA/RuvB/Holliday junction DNA complex formed the characteristic tripartite structure, with only one RuvA tetramer bound to one side of the Holliday junction, demonstrating the unexpected stability of this complex. The novel bent images of the complex revealed an intriguing morphological similarity to the structure of SV40 large T antigen, which belongs to the same AAA+ family as RuvB.     

Histone modifications in simian virus 40 and in nucleoprotein complexes containing supercoiled viral DNA.

Simian virus (SV40) nucleoprotein complexes containing circular supercoiled viral DNA were extracted from infected cells and purified by differential centrifugation. The protein content of these complexes was compared by electrophoresis on 15% acrylamide gels with the protein content of purified SV40 virions and with histones from virus-infected cells. The electrophoretic patterns of histones from each of the sources revealed several major differences. SV40 virions contained histones H3, H2B, H2A, and H4 but not H1. Nucleoprotein complexes and host cells contained all five major histone groups. Relative to cellular histones, virion and nucleoprotein complex histones were enriched 15 to 40% in histones H3 and H4. In addition to the major classes of histones, several subfractions of histones H1, H3, and H4 were observed in acrylamide gels of proteins from SV40 virions and viral nucleoprotein complexes. Acetate labeling experiments indicated that each subfraction of histones H3 and H4 had a different level of acetylation. The histones from SV40 virions and nucleoprotein complexes were acetylated to significantly higher levels than those of infected host cells. No apparent differences in phosphorylation of the major histone groups were observed.     

Regulation of mouse CYP1A1 gene expression by dioxin: requirement of two cis-acting elements during induction.

The mouse cytochrome P1450 (CYP1A1) gene is responsible for the metabolism of numerous carcinogens and toxic chemicals. Induction by the environmental contaminant tetrachlorodibenzo-p-dioxin (TCDD) requires a functional aromatic hydrocarbon (Ah) receptor. We examined the 5'-flanking region of the CYP1A1 gene in mouse hepatoma Hepa-1 wild-type cells and a mutant line having a defect in chromatin binding of the TCDD-receptor complex. We identified two cis-acting elements (distal, -1071 to -901 region; proximal, -245 to -50 region) required for constitutive and TCDD-inducible CYP1A1 gene expression. Three classes of DNA-protein complexes binding to the distal element were identified: class I, found only in the presence of TCDD and a functional Ah receptor, that was heat labile and not competed against by simian virus 40 (SV40) early promoter DNA; class II, consisting of at least three constitutive complexes that were heat stable and bound to SV40 DNA; and class III, composed of at least three constitutive complexes that were thermolabile and were not competed against by SV40 DNA. Essential contacts for these proteins were centered at -993 to -990 for the class I complex, -987, -986, or both for the class II complexes, and -938 to -927 for the class III complexes. The proximal element was absolutely essential for both constitutive and TCDD-inducible CYP1A1 gene expression, and at least two constitutive complexes bound to this region. These data are consistent with the proximal element that binds proteins being necessary but not sufficient for inducible gene expression; interaction of these proteins with those at the distal element was found to be required for full CYP1A1 induction by TCDD.     

Protein-induced bending of the simian virus 40 origin of replication

A 3.5 S protein, isolated from mammalian nuclei, specifically binds to DNA fragments containing the simian virus 40 (SV40) origin of replication. Two distinct nucleoprotein complexes are formed, a complex with high electrophoretic mobility carrying probably only one protein molecule, and a complex with reduced electrophoretic mobility carrying probably two protein molecules per DNA fragment. Band shift competition as well as methylation interference assays locate the binding site of the protein in the A + T-rich "late" region of the origin between SV40 nucleotides 13 and 35. The late origin binding (LOB) protein and T antigen bind simultaneously to adjacent sites in the origin. Using circularly permuted DNA fragments of identical lengths we show that the LOB protein induces pronounced bending of the origin fragment. The bending center maps at the 5' end of the adenine tract with one bound protein molecule and at the 3' end when two LOB proteins are bound to one origin fragment.