Action of Escherichia coli P1 restriction endonuclease on simian virus 40 DNA

The P1 restriction endonuclease (EcoP1) prepared from a P1 lysogen of Escherichia coli makes one double-strand break in simian virus (SV40) DNA. In the presence of cofactors S-adenosylmethionine and ATP the enzyme cleaves 70% of the closed circular SV40 DNA molecules once to produce unit-length linear molecules and renders the remaining 30% resistant to further cleavage. No molecules were found by electron microscopy or by gel electrophoresis that were cleaved more than once. It would appear that the double-strand break is made by two nearly simultaneous single-strand breaks, since no circular DNA molecules containing one single-strand break were found as intermediates during the cleavage reaction. The EcoP1 endonuclease-cleaved linear SV40 DNA molecules are not cleaved at a unique site, as shown by the generation of about 65% circular molecules after denaturation and renaturation. These EcoP1 endonuclease-cleaved, renatured circular molecules are resistant to further cleavage by EcoP1 endonuclease.The EcoP1 endonuclease cleavage sites on SV40 DNA were mapped relative to the partial denaturation map and to the EcoRI and HpaII restriction endonuclease cleavage sites. These maps suggest there are a minimum of four unique but widely spaced cleavage sites at 0.09, 0.19, 0.52, and 0.66 SV40 units relative to the EcoRI site. The frequency of cleavage at any particular site differs from that at another site. If S-adenosylmethionine is omitted from the enzyme reaction mix, SV40 DNA is cleaved into several fragments.An average of 4.6 ± 1 methyl groups are transferred to SV40 DNA from S-adenosylmethionine during the course of a normal reaction containing the cofactors. Under conditions which optimize this methylation, 7 ± 1 methyl groups can be transferred to DNA. This methylation protects most of the molecules from further cleavage. The methyl groups were mapped relative to the Hemophilus influenzae restriction endonuclease fragments. The A fragment receives three to four methyl groups and the B and G fragments each receive one to two methyl groups. These fragments correspond to those in which cleavage sites are located.     

Action of S1 nuclease on nicked circular simian virus 40 DNA.

SV40 DNA form II (FO II) containing on average more than one single strand nick per molecule was treated with S₁ nuclease. Linear duplex molecules of unit length (FO III) were generated at enzyme concentrations sufficient to achieve 95% hydrolysis of at least 100 times the amount of single-stranded DNA. Therefore, S₁ nuclease introduces under the described conditions only one double strand break per molecule despite the presence of several single strand nicks.     

Altered restriction endonuclease cleavage pattern of simian virus 40 DNA.

Three different groups of temperature-sensitive mutants of simian virus 40, isolated and characterized by Chou and Martin (J. Virol. 13:1101--1109, 1974), have been analyzed by using restriction endonucleases. Differences between the restriction endonuclease cleavage pattern of these mutants and that of the standard simian virus 40 strain have been mapped. These include the following observations: (i) tsD202 carries a defective HaeIII cleavage site at position 0.9 map units; (ii) tsB204 exhibits a defective HaIII site at position 0.21 and a defective HinIII site at 0.655 map units, and (iii) tsC219 carries a new HinIII site at position 0.15. We have isolated a few wild-type revertants from each of the temperature-sensitive mutant strains; each displays the endonuclease cleavage pattern of its parental temperature-sensitive strain.     

Self-annealing of 4 S strands from replicating simian virus 40 DNA

The nascent short strands (4 S) isolated from replicating Simian, virus 40 DNA hybridize specifically with denatured SV40 DNA and self-anneal extensively (70 to 92%) when incubated at 68 °C in 1 m-NaCl. Since complementary genetic sequences are present in the 4 S strands, both growing chains of SV40 DNA appear to be synthesized discontinuously at each replication fork.     

Salt-stable association of simian virus 40 capsid with simian virus 40 DNA

In 8 M CsCl, a fraction of the wild-type previrions and tsB228 nucleoprotein complexes lose their core histones but retain their capsid. These histone-depleted complexes appear in the electron microscope as a protein shell attached to supercoiled DNA. Consistent with this result, we find that in 1 M NaCl, the wild-type previrions dissociate into two populations of nucleoprotein complexes. One population sediments between 50 and 140 S and morphologically resembles the shell-DNA complexes isolated in CsCl gradients. The other population is comprised primarily of nucleoproteins which sediment at 40 S.     

Loss of supercoiled viral DNA after infection of permissive cells by simian virus 40.

During infection of BS-C-1 cells (a permissive line of monkey kidney cells) with simian virus 40, there was a loss of infecting viral supercoiled (component I) DNA molecules which was partially inhibited by cycloheximide. The kinetics of loss of component I in the presence of cycloheximide suggested that the loss was the result of at least two activities acting on the DNA, one cycloheximide sensitive and the second cycloheximide insensitive. Nucleotides from degraded infecting viral DNA were reincorporated into cell DNA that was synthesized during the infection.     

DNase I sensitivity of integrated simian virus 40 DNA.

We undertook an analysis of integrated simian virus 40 (SV40) DNA to learn whether the DNase I-sensitive region is retained in the integrated array of mouse transformants. Our results indicate that full-length integrated SV40 chromatin retains a DNase I-hypersensitive region at the same point as in nonintegrated SV40 chromatin. Thus, the lack of a DNase I-hypersensitive region is not likely to be the reason for nonpermissivity of SV40 in mouse cells. In addition, results reported here indicate that a deletion of about 200 base pairs of DNA in the region of the DNase I-hypersensitive site severely reduces the sensitivity of integrated SV40 chromatin. This result is similar to a previously reported result obtained with deletion mutants of SV40 analyzed in the lytic cycle. It is the first report of a DNA lesion affecting DNase I hypersensitivity of a mammalian chromosome.     

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.     

Specificity of the S1 nuclease from Aspergillus oryzae.

Conditions are described for digesting single-stranded DNA by S1 nuclease without introducing breaks in double-stranded DNA. The enzyme is inhibited by low concentrations of various compounds of phosphate. Under certain conditions S1 nuclease cleaves the strand opposite a nick in bacteriophage T5 DNA; under other conditions, the enzyme cleaves a loop in one strand of heteroduplex lambdaDNA while leaving the opposite strand intact. S1 nuclease makes many single strand breaks in ultraviolet-irradiated duplex lambdaDNA. Superhelical DNA of phiX174 (Form I) is converted first to a relaxed circular molecule (Form II), and then to a linear molecule (Form III) by cleavage at one site per molecule. Since the cleavage occurs at many sites in the population of molecules, the partially single-stranded regions in phiX174 superhelical DNA are not determined by specific nucleotide sequences.     

Amino acid sequence of nuclease S1 from Aspergillus oryzae

The amino acid sequence of nuclease S1, a nuclease which cleaves both single-stranded DNA and RNA, from Aspergillus oryzae was determined. Reduced and S-carboxymethylated or S-aminoethylated nuclease S1 was digested with Achromobacter protease I, Staphylococcus aureus V8 protease, or endoproteinase Asp-N. Peptides thus obtained were purified by reverse-phase high-performance liquid chromatography and sequenced, and the complete primary structure was established. Nuclease S1 consists of a single peptide chain of 267 amino acid residues bearing N-glycosylated Asns 92 and 228. Five half-cystine residues are present at positions 25, 72, 80, 85, and 216, and the latter four residues are implicated in the formation of disulfide bonds by analogy with those in nuclease P1. Two short stretches of sequences involving His 60 and His 125 are shown to be identical with those involving active site His 119 in bovine ribonuclease A and active-site His 134 in porcine deoxyribonuclease I, respectively.     

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.     

DNA primase-DNA polymerase alpha from simian cells: sequence specificity of initiation sites on simian virus 40 DNA.

Unique single-stranded regions of simian virus 40 DNA, phage M13 virion DNA, and several homopolymers were used as templates for the synthesis of (p)ppRNA-DNA chains by CV-1 cell DNA primase-DNA polymerase alpha. Intact RNA primers, specifically labeled with an RNA capping enzyme, were typically 6 to 8 ribonucleotides long, although their lengths ranged from 1 to 9 bases. The fraction of intact RNA primers 1 to 4 ribonucleotides long was 14 to 73%, depending on the template used. RNA primer length varied among primers initiated at the same nucleotide, as well as with primers initiated at different sites. Thus, the size of an RNA primer depended on template sequence. Initiation sites were identified by mapping 5' ends of nascent RNA-DNA chains on the template sequence, identifying the 5'-terminal ribonucleotide, and partially sequencing one RNA primer. A total of 56 initiation events were identified on simian virus 40 DNA, an average of 1 every 16 bases. Some sites were preferred over others. A consensus sequence for initiation sites consisted of either 3'-dCTTT or 3'-dCCC centered within 7 to 25 pyrimidine-rich residues; the 5' ends of RNA primers were complementary to the dT or dC. High ATP/GTP ratios promoted initiation of RNA primer synthesis at 3'-dCTTT sites, whereas low ATP/GTP ratios promoted initiation at 3'-dCCC sites. Similarly, polydeoxythymidylic acid and polydeoxycytidylic acid were the only effective homopolymer templates. Thus, both template sequence and ribonucleoside triphosphate concentrations determine which initiation sites are used by DNA primase-DNA polymerase alpha. Remarkably, initiation sites selected in vitro were strikingly different from initiation sites selected during simian virus 40 DNA replication in vivo.     

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.     

Class I major histocompatibility proteins are an essential component of the simian virus 40 receptor.

The class I molecules encoded by the major histocompatibility complex (MHC) present endogenously synthesized antigenic peptide fragments to cytotoxic T lymphocytes. We show here that these proteins are an essential component of the cell surface receptor for simian virus 40 (SV40). First, SV40 binding to cells can be blocked by two monoclonal antibodies against class I human lymphocyte antigen (HLA) proteins but not by monoclonal antibodies specific for other cell surface proteins. Second, SV40 does not bind to cells of two different human lymphoblastoid cell lines which do not express surface class I MHC proteins because of genetic defects in the beta 2-microglobulin gene in one line and in the HLA complex in the other. Transfection of these cell lines with cloned genes for beta 2-microglobulin and HLA-B8, respectively, restored expression of their surface class I MHC proteins and resulted in concomitant SV40 binding. Finally, SV40 binds to purified HLA proteins in vitro and selectively binds to class I MHC proteins in a cell surface extract.     

cis-active elements from mouse chromosomal DNA suppress simian virus 40 DNA replication.

Simian virus 40 (SV40)-containing DNA was rescued after the fusion of SV40-transformed VLM cells with permissive COS1 monkey cells and cloned, and prototype plasmid clones were characterized. A 2-kilobase mouse DNA fragment fused with the rescued SV40 DNA, and derived from mouse DNA flanking the single insert of SV40 DNA in VLM cells, was sequenced. Insertion of the intact rescued mouse sequence, or two nonoverlapping fragments of it, into wild-type SV40 plasmid DNA suppressed replication of the plasmid in TC7 monkey cells, although the plasmids expressed replication-competent T antigen. Rat cells were transformed with linearized wild-type SV40 plasmid DNA with or without fragments of the mouse DNA in cis. Although all of the rat cell lines expressed approximately equal amounts of T antigen and p53, transformants carrying SV40 DNA linked to either of the same two replication suppressor fragments produced significantly less free SV40 DNA after fusion with permissive cells than those transformed by SV40 DNA without a cellular insert or with a cellular insert lacking suppressor activity. The results suggest that two independent segments of cellular DNA act in cis to suppress SV40 replication in vivo, either as a plasmid or integrated in chromosomal DNA.     

DNA binding properties of a mutant T antigen from the simian virus 40-transformed human cell line simian virus 80

We investigated whether the T antigen of the simian virus 40-transformed human cell line simian virus 80 ( SV80 ) specifically recognizes DNA sequences of its own template, i.e, the viral sequences integrated in the SV80 cellular genome. In vitro DNA binding experiments clearly indicated that, in contrast to wild-type T antigen, SV80 T antigen does not specifically bind to sites on the integrated viral DNA in SV80 cells.