Chemical modification of S-adenosylhomocysteinase by a water-soluble carbodiimide

S-Adenosylhomocysteinase (EC 3.3.1.1) from rat liver is inactivated by 1-cyclohexyl-3-(2-morpholinoethyl)carbodiimide metho-p-toluenesulfonate (CMC) in a pseudo-first-order fashion. The rate of inactivation is linearly related to the concentration of the reagent, and a second-order rate constant of 4.94 +/- 0.27 M-1 min-1 is obtained at pH 5.5 and 25 degrees C. The inactivation does not involve change in the quaternary structure of the enzyme nor modification or release of the enzyme-bound NAD. Lack of modification at tyrosine, serine, cysteine, histidine, and lysine residues and the fact that the inactivation is favored at low pH suggest that the inactivation is caused by the modification of a carboxyl group. Statistical analysis of the relationship between the residual enzyme activity and the extent of modification, and comparison of the number of residues modified in the presence and absence of the substrate adenosine show that, among four reactive residues per enzyme subunit, only one residue which reacts more rapidly with the reagent than the rest is critical for activity. The CMC-modified enzyme binds adenosine and S-adenosylhomocysteine and is able to oxidize the 3' hydroxyl of these substrates, but apparently fails to catalyze the abstraction of the 4' proton of adenosine.     

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.     

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.     

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.     

DNA synthesis by partially purified replicating simian virus 40 chromosomes.

We have partially purified replicating simian virus 40 (SV40) chromosomes in a form which allows continued DNA synthesis in vitro. We first prepare a soluble DNA-synthesizing system from SV40-infected monkey cells and then sediment the components through a neutral sucrose gradient of extremely low ionic strength. Replicating SV40 chromosomes isolated from such gradients are capable of continuing DNA synthesis in vitro in the same manner as two crude subnuclear systems we have previously described (4). This indicates that the enzymes and other proteins required for in vitro DNA synthesis are bound to the replicating chromosomes.     

Perturbations in simian virus 40 DNA synthesis by ultraviolet light.

Perturbations of Simian Virus 40 (SV40) DNA replication by ultraviolet (UV) light during the lytic cycle in permissive monkey CV-1 cells resemble those seen in host cell DNA replication. Formation of Form I DNA molecules (i.e. completion of SV40 DNA synthesis) was more sensitive to UV irradiation than synthesis of replicative intermediates or Form II molecules, consistent with inhibition of DNA chain elongation. The observed amounts of [3H]thymidine incorporated in UV-irradiated molecules could be predicted on the assumption that pyrimidine dimers are responsible for blocking nascent DNA strand growth. The relative proportion of labeled Form I molecules in UV-irradiated cultures rapidly increased to near-control values with incubation after 20 or 40 J/m2 of light (0.9--1.0 or 1.8--2.0 dimers per SV40 genome, respectively). This rapid increase and the failure of Form II molecules to accumulate suggest that SV40 growing forks can rapidly bypass many dimers. Form II molecules formed after UV irradiation were not converted to linear (Form III) molecules by the dimer-specific T4 endonuclease V, suggesting either that there are no gaps opposite dimers in these molecules or that T4 endonuclease V cannot use Form II molecules as substrates.     

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.     

A malignant astrocytoma containing simian virus 40 DNA in a macaque infected with simian immunodeficiency virus

Abstract: Polyomaviruses have proven oncogenicity in nonhost experimental animals; however, studies concerning the association between human brain tumors and simian and human polyomaviruses have yielded inconclusive results. We examined the relationship of SV40 to a malignant astrocytoma found in the right frontal lobe of a pigtail macaque (Macaca nemestrina) infected with simian immunodeficiency virus (SIV). Consistent with the histologic diagnosis, the tumor was immunoreactive with antibodies to S-100 protein, vimentin, and glial fibrillary acidic protein, but negative for neurofilament protein, synaptophysin, neuron-specific enolase, and chromogranin A. At the time of SIV inoculation, the animal was seropositive for SV40. Polymerase chain reaction assay of tumor DNA, but not normal brain DNA, yielded a 300 base-pair fragment corresponding to the carboxy-terminal coding region (C-terminus) of the large T antigen gene of SV40, suggesting an association with the tumor.     

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.     

Initiation of simian virus 40 DNA synthesis in vitro.

Simian virus 40 (SV40) T antigen can efficiently initiate SV40 origin-dependent DNA synthesis in crude extracts of HeLa cells. Therefore, initiation of SV40 DNA synthesis can be analyzed in detail. We present evidence that antibodies which neutralize proliferating cell nuclear antigen (PCNA) inhibit but do not abolish pulse-labeling of nascent DNA. The lengths of DNA products formed after a 5-s pulse in the absence and presence of anti-PCNA serum averaged 150 and 34 nucleotides, respectively. The small DNAs formed in the presence of anti-PCNA serum underwent little or no increase in size during further incubation periods. The addition of PCNA to reaction mixtures inhibited with anti-PCNA serum largely reversed the inhibitory effect of the antiserum. The small nascent DNAs formed in the presence or absence of anti-PCNA serum products arose from the replication of lagging strands. These results suggest that a PCNA-dependent elongation reaction participates in the synthesis of lagging strands as well as leading strands. We also present evidence that in crude extracts of HeLa cells, DNA synthesis generally does not initiate within the core origin. Initiation of DNA synthesis outside of a genetically defined origin region has not been previously described in a eukaryotic replication system but appears to be a common feature of initiation events in many prokaryotic organisms. Additional results presented indicate that in the absence of nucleoside triphosphates other than ATP, the preinitiation complex remains within or close to the SV40 origin.     

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.     

Association of simian virus 40 T antigen with replicating nucleoprotein complexes of simian virus 40.

An immunoprecipitation assay was established for simian virus 40 T-antigen-bound nucleoprotein complexes by means of precipitation with sera from hamsters bearing simian virus 40-induced tumors. About 80% of simian virus 40 replicating nucleoprotein complexes in various stages of replication were immunoprecipitated. In contrast, less than 21% of mature nucleoprotein complexes were immunoprecipitated. Pulse-chase experiments showed that T antigen was lost from most of the nucleoprotein complexes concurrently with completion of DNA replication. T antigen induced by dl-940, a mutant with a deletion in the region coding for small T antigen, was also associated with most of the replicating nucleoprotein complexes. Once bound with replicating nucleoprotein complexes at the permissive temperature, thermolabile T antigen induced by tsA900 remained associated with the complexes during elongation of the replicating DNA chain at the restrictive temperature. These results suggest that simian virus 40 T antigen (probably large T antigen) associates with nucleoprotein complexes at or before initiation of DNA replication and that the majority of the T antigen dissociates from the nucleoprotein complexes simultaneously with completion of DNA replication.     

Selectivity of interferon action in simian virus 40-transformed cells superinfected with simian virus 40.

Treatment of African green monkey kidney CV-1 cells with human alpha interferons before infection with simian virus 40 (SV40) inhibited the accumulation of SV40 mRNAs and SV40 T-antigen (Tag). This inhibition persisted as long as the interferons were present in the medium. SV40-transformed human SV80 cells and mouse SV3T3-38 cells express Tag, and interferon treatment of these cells did not affect this expression. SV80 and SV3T3-38 cells which had been exposed to interferons were infected with a viable SV40 deletion mutant (SV40 dl1263) that codes for a truncated Tag. Exposure to interferons inhibited the accumulation of the truncated Tag (specified by the infecting virus) but had no significant effect on the accumulation of the endogenous Tag (specified by the SV40 DNA integrated into the cellular genome). The level of Tag in SV40-transformed mouse SV101 cells was not significantly decreased by interferon treatment. SV40 was rescued from SV101 cells and used to infect interferon-treated and control African green monkey kidney Vero cells. Tag accumulation was inhibited in the cells which had been treated with interferons before infection. Our data demonstrate that even within the same cell the interferon system can discriminate between expression of a gene in the SV40 viral genome and expression of the same gene integrated into a host chromosome.     

DNA polymerase activities in growing cells infected with simian virus 40.

Growing CV1 cells were infected with simian virus 40 (SV40), and the levels of DNA polymerases-alpha, -beta, and -gamma were analyzed in the cytoplasm, nuclear Triton wash, and nucleus. In the cytoplasmic fraction, the amount of alpha-, beta-, or gamma-polymerase remained unaltered after SV40 infection. The activity of DNA polymerase-alpha increased five- to sixfold in the nuclear Triton wash and threefold in the nuclei and then remained enhanced only inside the nuclei. That of DNA polymerases-beta and gamma increased mostly in the nuclei after infection. These results suggest that DNA polymerase-alpha could be the major enzyme involved in SV40 DNA replication.     

Specific association of simian virus 40 tumor antigen with simian virus 40 chromatin

Simian virus 40 tumor antigen (SV40 T antigen) was bound to both replicating and fully replicated SV40 chromatin extracted with a low-salt buffer from the nuclei of infected cells, and at least a part of the association was tight specific. T antigen cosedimented on sucrose gradients with SV40 chromatin, and T antigen-chromatin complexes could be precipitated from the nuclear extract specifically with anti-T serum. From 10 to 20% of viral DNA labeled to steady state with [3H]thymidine for 12 h late in infection or 40 to 50% of replicating viral DNA pulse-labeled for 5 min was associated with T antigen in such immunoprecipitates. After reaction with antibody, most of the T antigen-chromatin complex was stable to washing with 0.5 M NaCl, but only about 20% of the DNA label remained in the precipitate after washing with 0.5 M NaCl-0.4% Sarkosyl. This tightly bound class of T antigen was associated preferentially with a subfraction of pulse-labeled replicating DNA which comigrated with an SV40 form I marker. A tight binding site for T antigen was identified tentatively by removing the histones with dextran sulfate and heparin from immunoprecipitated chromatin labeled with [32P]phosphate to steady state and then digesting the DNA with restriction endonucleases HinfI and HpaII. The site was within the fragment spanning the origin of replication, 0.641 to 0.725 on the SV40 map.