Polyoma DNA Synthesis Research Articles

Nitropyrenes are inducers of polyoma viral DNA synthesis

The biological activity of a series of nitopyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a st-a mutant of polyoma virus. Concentrations of 10–30 μg/ml of 1-nitropyrene (1-NP) induced viral replications, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 μ/ml) to the culture medium. The response was less than that obtained with 0.1 μg/ml of the activated metabolite of benzo[ a]pyrene (BP), BP trans-7,8-dihydrodiol-9,10 epoxide (anti) (BPDE). A series of di-, tri-, and tetra-nitropyrenes were also found to induce polyoma DNA replicatin, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5–2.0 μg/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 μg/ml inidcated that this compound was approximately equipotent with BPDE for induction of polyoma DNA synthesis. Studies of drug metabolism, DNA binding and DNA adduct formation indicate that 1,6-DNP is metabolized in this cell line, binds to DNA, and forms stable adducts. The level of DNA modifications seen with, 1,6-DNP is higher than that observed under comparable conditions with an equivalent dose of BPDE. These findings provide additional evidence that the nitropyrene class of compounds can exert biological effects in mammalian cells, and that the dinitropyrenes are more potent than 1-NP.

Further characterization of the phenotype of ts20, a DNA ts mutant of BALB/3T3 cells

Ts20 is a temperature-sensitive mutant cell line derived from BALB/3T3 cells that is blocked at a step in DNA synthesis involving chain elongation. Following a shift from 33 ° to 39 °C, mutant cells lost ability to grow or form colonies. When mutant cells were infected with polyomavirus, both cell and virus DNA synthesis were inhibited at the restrictive temperature of 39 °C. When cell extracts from wild-type cells were added in vitro to lysed infected mutant cells that had been incubated in vivo at 39 °C for expression of the mutation, cell DNA synthesis was increased 3-fold (similar to the effect in uninfected mutant cells), whereas virus DNA synthesis was increased only 60%. With harsher lysis conditions, the effect of added extract on virus DNA synthesis was greater, although baseline DNA synthesis (prior to addition of extracts) was much lower. Analysis by alkaline sucrose gradients showed that the addition of cell extract converted small cellular DNA molecules into larger ones, while it increased the synthesis of small virus DNA molecules rather than completed genomes. Analysis of cytosol extracts (in which the activity stimulating DNA synthesis resides) showed that DNA topo-isomerase I activity was more heat-labile when assayed in mutant extracts compared to wild-type extracts. In contrast, cytosol DNA polymerase activity was equally heat-labile in mutant and wild-type extract. This suggested the factor in extract was likely associated with the activity of DNA topo-isomerase I. Analysis of virus DNA synthesized in vitro in restricted mutant cells by gel electrophoresis and fluorography showed an accumulation of topo-isomers migrating between form I and II. These topo-isomers, thought to be a manifestation of the ts defect, did not disappear when extract from wild-type cells was added back in vitro or when mutant cells were shifted back to permissive temperature prior to lysis for in vitro synthesis. The results indicate that polyoma DNA synthesis and cell DNA synthesis differ in their response to the mutant gene product in ts20, although both are inhibited at a step early in DNA chain elongation that may involve DNA topo-isomerase I.

Inhibition of polyoma DNA synthesis by base pair substitutions at the replication origin.

The effect of base pair substitutions on the function of the polyoma virus origin of DNA replication was studied. The mutations were all C-G to T-A transitions, induced by bisulfite treatment of recombinant DNA molecules. The mutagenesis was directed to short single-stranded gaps in duplex DNA, or to loops in heteroduplex molecules. Modification of a 34 base pair sequence of dyad symmetry led to cis-acting inhibition of viral DNA synthesis, ranging from slight defects to total inactivation. One of the mutants was temperature sensitive. Mutants with base changes in an adjacent DNA segment, including an 18 base pair long purine-pyrimidine tract, had similar, but less severe, deficiences. In contrast to the effect of mutations in the homologous region of the simian virus 40 genome, there was no strict relationship between mutation of the putative large T-antigen-binding base sequence GPuGGC and defective viral DNA synthesis.

Benzo[a]pyrene induction of extrachromosomal viral DNA synthesis in rat cells transformed by polyoma virus.

The effect of the carcinogen benzo[a]pyrene (BP) on the production of specific extrachromosomal DNAs has been studied in transformed rat cells containing integrated polyoma virus DNA. Exposure of cells previously transformed by the polyoma virus mutant ts-a, which codes for a temperature sensitive T antigen, to a non-toxic dose of BP (0.25 micrograms/ml) enhanced the production of free polyoma DNA at the population level. This occurred at 33 degrees C (the permissive temperature) but not at 39 degrees C, indicating that the BP effect was dependent on the function of the T antigen of polyoma virus. In the same cell system, BP did not induce the production of extrachromosomal copies of two endogenous rat retrovirus genomes, the 30S RNA virus and rat leukemia virus. Thus, the effects on integrated polyoma DNA are quite specific and do not reflect a generalized increase in asynchronous DNA replication and excision. Further studies utilizing a carcinogenic metabolite of BP, benzo[a]pyrene trans-7,8-dihydrodiol-9,10-epoxide (anti) (BPDE), on a rat cell line transformed by wild type (WT) polyoma virus demonstrate that the enhancement of polyoma DNA synthesis persists for at least 5 days after a single exposure to BPDE, despite the rapid decay of this compound. In addition, enhanced synthesis of polyoma DNA can be induced by fusion of normal rat fibroblasts previously exposed to BPDE with polyoma transformed rat fibroblasts not exposed to BPDE. It appears, therefore, that the effects we have observed are not due solely to direct carcinogen damage at the level of the integrated polyoma virus DNA, but may instead involve the induction of cellular factor(s) that act indirectly to enhance asynchronous replication of polyoma DNA.

Isolation and characterization of replication forks from discrete regions of the polyoma genome.

Synthesis of polyoma DNA in nuclei isolated from virus-infected 3T6 mouse fibroblasts leads to the selective labeling of replicative intermediates. Digestion of these replicative intermediates with the restriction endonuclease HpaII resulted in three highly labeled heterogeneous species in addition to the expected full-length fragments. These three species migrated more slowly in agarose than did any of the full-length restriction fragments and were shown to represent families of replication forks by criteria of sensitivity to S1 nuclease, kinetics of labeling both in vitro and in vivo, electron microscopy, and migration behavior during agarose gel electrophoresis. Subsequent digestion with other restriction enzymes showed that the two largest of the three fork bands originated from HpaII fragments 1 and 2. These fragments flank the putative terminus located 180 degrees relative to the origin. The third fork-containing band was less labeled and was derived from fragment 3, which is juxtaposed to the replication origin on the side corresponding to late transcription. A two-dimensional gel system revealed the presence of a fourth fork band, derived from fragment 4, that was obscured by full-length fragments 1 and 2 in the single-dimension electrophoresis. Resolution of the fork families revealed multiple discrete species within the major bands, implying the existence of stops or hesitations during replication of a given region of the genome. This conclusion is consistent with the presence of multiple species upon electrophoresis of the fork bands under denaturing conditions.

Deoxyuridine triphosphate pools after polyoma virus infection.

The synthesis of polyoma DNA in virus-infected 3T6 mouse fibroblasts is discontinuous with the intermediate formation of short Okazaki fragments. Hydroxyurea, an inhibitor of the enzyme ribonucleotide reductase, inhibits polyoma DNA synthesis, as measured by incorporation of radioactive thymidine. In the inhibited state, almost all incorporation occurs into short fragments. We investigated to what extent formation of short DNA fragments might be the result of incorporation of deoxyuridine triphosphate (dUTP) into DNA, followed by excision and repair reactions. We devised a sensitive enzymatic method for measuring dUTP in cell extracts which allows the determination of the dUTP pool when this pool amounts to between 0.1 and 2% of the dTTP pool. No dUTP was detected in growing mouse fibroblasts. After infection with polyoma virus cell extracts contained 0.4% dUTP (of dTTP) at the peak of DNA synthesis. Addition of hydroxyurea at this point led to a disappearance of dUTP. We conclude that dUTP incorporation can contribute only minimally to the generation of short fragments during polyoma DNA synthesis.

Replication of polyoma DNA in isolated nuclei: analysis of replication fork movement

The movement of replication forks during polyoma DNA synthesis in isolated nuclei was analyzed by digesting newly synthesized DNA with the restriction endonuclease HpaII which cleaves polyoma DNA into eight unique fragments. The terminus of in vitro DNA synthesis was identified by cleaving newly completed molecules with HpaII. The distribution of label in the restriction fragments showed that the in vitro DNA synthesis was bidirectional and had the normal terminus of replication. Analysis of replicative intermediates pulse-labeled in vitro further suggested that DNA synthesis in isolated nuclei is an ordered process similar to replication in intact cells. Replication forks moved with a constant rate from the origin towards the terminus of replication. The nonlinear course of the DNA synthesis reaction in the isolated nuclei seems to result from the random inactivation of replication forks rather than a decrease in the rate of fork movement. During the in vitro synthesis a replication fork could maximally synthesize a DNA chain about 1,000 nucleotides long. The results suggest that some replication forks might be initiated in vitro at the origin of replication.

Polyoma DNA synthesis in isolated nuclei: evidence for defective replication forks

Nuclei, isolated from polyoma virus-infected mouse 3T6 cells, were incubated under condtions suitable for polyoma DNA synthesis. By using electrom microscopy and standard regression statistics, it was shown that replication is mainly unidirectional in a large number of molecules, indicating the presence of inactive replication forks. The replication forks were inactivated randomly, and the defect seemed to be present from the beginning of the in vitro incubation.

Replication of polyoma DNA in isolated nuclei: VII. Initiator RNA synthesis during nucleotide depletion

Earlier experiments demonstrated that the Okazaki fragments synthesized during discontinuous polyoma DNA synthesis in isolated nuclei at their 5′ ends contained structural elements consisting of polyribonucleotides starting with ATP or GTP (Reichard et al., 1974) . These structures could be released by digestion with pancreatic DNAase and were named initiator RNA. They consist of a large family of polyribonucleotides differing in base sequence but having a common size of about a decanucleotide. We now demonstrate that limitation of DNA synthesis by low concentrations of deoxyribonucleoside triphosphates in parallel limits the synthesis of initiator RNA. This is additional evidence for the primer function of initiator RNA. When ribonucleoside triphosphates other than ATP were deleted from the incubation medium only a small decrease of DNA and initiator RNA synthesis occurred. Under those conditions deoxyribonucleotides substituted for ribonucleotides and were incorporated internally into the primer. From this result as well as the insensitivity of initiator RNA synthesis to α-amanitin (Reichard & Eliasson, 1979) we suggest that a mammalian counterpart to primase, the dnaG gene product of Escherichia coli (Rowen & Kornberg, 1978 a) , catalyzes the synthesis of initiator RNA.

Primase initiates Okazaki pieces during polyoma DNA synthesis

SHORT fragments (Okazaki pieces) are formed as intermediates during elongation of nascent DNA strands1. In isolated nuclei from polyoma-virus infected 3T6 cells Okazaki pieces are initiated by a short RNA primer (initiator RNA, iRNA) with the approximate size of a decanucleotide2. The nature of the polymerase responsible for primer synthesis is not known, but high concentrations of α-amanitin, a drug which inhibits both RNA polymerase II (ref. 3) and III (ref. 4), have no effect on primer formation (unpublished results). We now present evidence that iRNA is not synthesised by any of the known RNA polymerases, but that, instead, a mammalian enzyme is involved which in its action corresponds to the dnaG gene product of E. coli. The bacterial enzyme was named primase5. It participates in the initiation of DNA strands of some single-stranded bacteriophages and has been suggested to be involved in the synthesis of bacterial Okazaki pieces6,7. One of the distinguishing features of the E. coli enzyme is its ability to use both ribonucleotides and deoxyribonucleotides for primer synthesis5,8.

Formation of Okazaki fragments in polyoma DNA synthesis caused by misincorporation of uracil

When dUTP replaced dTTP during polyoma DNA replication in isolated cell nuclei, radioactivity from labeled deoxynucleoside triphosphates was almost exclusively recovered in very short Okazaki fragments and incorporation ceased after a short time. Addition of uracil, a known inhibitor of the enzyme uracil-DNA glycosidase (Lindahl et al., 1977), increased total synthesis and shifted the incorporation to longer progeny strands. The presence of as little as 2.5% of dUTP in a dTTP-containing system gave a distinct increase in isotope incorporation into Okazaki pieces accompanied by a corresponding decrease in longer strands. This effect was reversed completely by uracil. The short strands formed from dUTP could be chased efficiently into long strands. Our results suggest that dUTP can be incorporated in place of dTTP into polyoma DNA, and that polyoma-infected nuclei, similar to E. coli (Tye et al., 1977), contain an excision-repair system which by removal of uracil causes strand breakage and under certain circumstances may contribute to the formation of Okazaki fragments.

2'-Deoxy-2'-azidocytidine inhibits the initiation of polyoma DNA synthesis.

2'-Deoxy-2'-azidocytidine, a nucleoside analogue, inhibits polyoma DNA synthesis. The inhibition results in a 5.5-fold decrease in the amount of replicating intermediates, as detected by electron microscopy. The results indicate that replication is inhibited at an early step, possibly in the initiation of new rounds of replication. In contrast, the effect on the elongation of DNA chains appears to be less pronounced. As a result of the inhibition, polyoma DNA molecules at an early stage of replication accumulate in the nuclei. Upon incubation of such nuclei, DNA synthesis is induced in these molecules. The replication starts from the normal origin and elongation proceeds bidirectionally at a rate close to that observed during in vivo conditions. Analysis of polyoma DNA replication in nuclei isolated from control cells indicates that both initiation and termination of replication are impaired under in vitro conditions.

In vitro polyoma DNA synthesis: Asymmetry of short DNA chains

The kinetics of annealing of the separated strands of the polyoma DNA Hpa II restriction fragments 1 and 2 to an excess of purified short DNA chains isolated from in vitro pulse-labeled replicating polyoma DNA were determined. The results indicate that for each growing fork, the DNA strand which must grow discontinuously is represented about 4 times as frequently in the population of short DNA chains as the strand which could replicate continuously. In addition, the absolute concentration of short DNA chains in the two growing forks is approximately the same. The average size of the short DNA chains from the continuous strand was shown to be very similar to that of the short DNA chains from the discontinuous strand. We conclude that polyoma DNA replication in vitro proceeds by a predominantly semi-discontinuous mechanism.

Polyoma-induced stimulation of cellular RNA synthesis is paralleled by changed expression of the viral genome

We studied synthesis of viral and cellular RNA in the presence and absence of 5-fluorodeoxyuridine (FdU, an inhibitor of DNA synthesis) during lytic infection with polyoma virus in confluent, primary mouse kidney cell cultures. In the presence of FdU, synthesis of early 19S polyoma mRNA and of polyoma tumor (T)-antigen, i.e. expression of the early viral gene, is rapidly followed by a mitogenic reaction of the host cell; it leads to an increase of 30 +/- 5% in cellular, mainly 28S and 18S rRNA, followed by activation of the cellular DNA-synthesizing apparatus. Polyoma-induced cellular RNA synthesis is paralleled by increased production of early 19S mRNA and begin of expression of the late viral genes, leading to synthesis of small amounts of late 19S and 16S mRNAs. Changed expression of the viral genome occurs in the absence of detectable synthesis of polyoma DNA I. Infection in the absence of FdU induces the same sequence of events; it is followed, however, by duplication of the mouse cell chromatin (S-phase) and production of progeny virus.

Replication of polyoma DNA accumulation of early replicative intermediates during hydroxyurea inhibition

Abstract Inhibition of polyoma DNA synthesis by hydroxyurea appears to affect the initiation of new rounds of DNA synthesis to a lesser extent than chain elongation. Viral DNA synthesized in hydroxyurea-treated 3T6 cultures showed an accumulation of early replicative intermediates, in which the progeny strands were shorter than 20% of the genome length. No such accumulation was found for polyoma DNA replicating in control cells, where the replicative intermediates were evenly distributed at all stages of replication.

Polyoma and cell DNA synthesis in mouse L cells temperature sensitive for the replication of cell DNA

Polyoma (Py) virus multiplies, at 34 and 38.5 C, in wild-type (WT-4) and in ts A1S9 mouse L cells, which are temperature sensitive for growth and for DNA replication (R. Sheinin, 1976; L. H. Thompson et al., 1970). De novo synthesis of double-stranded, fully covalently closed Py DNA has been shown to proceed by semiconservative replication in WT-4 and ts A1S9 cells at the permissive and nonpermissive temperatures. Cell DNA is made late during infection, by both cell types and at both temperatures. Semiconservative replication of cell DNA proceeds in Py-infected WT-4 cells incubated at 34 or at 38.5 C and in Py-infected ts A1S9 cells incubated at 34 C. In virus-infected ts A1S9 cells incubated at 38.5 C, cell DNA synthesis appears to proceed almost entirely by a process analogous to repair replication. The inability of ts A1S9 cells to produce large-molecular-weight chromosomal DNA strands, at 38.5 C, by the normal mechanism is not overcome by Py infection.

Temperature-sensitive mutants of Balb/3T3 cells: Description of a mutant affected in cellular and polyoma virus DNA synthesis

A temperature-sensitive Dna— mutant (ts-2) of the mouse cell Balb/3T3 is characterized. Studies with synchronized cells indicate that the defect is in DNA synthesis itself, rather than in progress toward its initiation. ts-2 supports polyoma DNA synthesis after infection at 33°C but not at 38°C. Viral DNA synthesis begun at 33°C is inhibited upon shift to 38°C. A procedure is proposed by which viral DNA synthesis can be used to distinguish different classes of cell Dna— mutants.

In vitro polyoma DNA synthesis: Self-annealing properties of short DNA chains

Short DNA chains, isolated from in vitro pulse-labeled replicating polyoma DNA, exhibit some degree of self-complementarity (28% resistance to S1 nuclease after self-annealing to plateau levels). This level of self-annealing is not increased if short DNA chains present as free single-stranded DNA after extraction are included in the hybridization, excluding a selective loss of chains from one side of the growing fork and supporting a semi-discontinuous mode of chain growth. This mode also applies to restricted synthesis conditions under which a relative excess of short chains is made, since no increase in the self-annealing of such short chains is observed. The self-annealing that can be measured is higher for the faster sedimenting portion (46%) of the short DNA chains than for the slower sedimenting portion (18.5%), indicating that it is most likely due to contaminating continuously growing strands from the other side of the fork. High self-annealing values (up to 60%) are obtained if virus stocks generating defective DNA are used for infection. Restriction endonuclease (HpaII) characterization of such DNA shows evidence for the presence of multiple origins of replication. One of several possible mechanisms is discussed by which replicating defective DNA can generate self-complementary short chains despite a semi-discontinuous mode of replication.

Interactions of Polyoma and Mouse DNAs III. Mechanism of Polyoma Pseudovirion Formation

In primary mouse kidney cell cultures infected with polyoma virus, the processes leading to virion and pseudovirion formation were studied. By photometric DNA quantitation, we followed the kinetics of mouse and polyoma DNA synthesis and the formation of low-molecular-weight fragmented mouse DNA (mouse f-DNA). Virus was harvested at different times and analyzed for its proportion of pseudovirions. The following correlations between the intracellular events and the production of virions and pseudovirions were found. (i) Syntheses of cellular and viral DNA were closely linked, both in time and in rates of synthesis. (ii) An increase of mouse f-DNA could only be detected several hours after the onset of mouse and polyoma DNA replication; its formation coincided in time with the appearance of progeny virus. (iii) The proportion of pseudovirions was not dependent on the amount of mouse f-DNA formed, but seemed to be inversely related to the amount of viral DNA synthesized. This was borne out by experiments in which DNA synthesis was partially inhibited by mitomycin C or after a synchronized onset of DNA replication. Under these conditions, virus preparations with a two- to threefold increased proportion of pseudovirions were obtained as compared with those from uninhibited cultures. Virus isolated from the remaining monolayer always had a higher proportion of pseudovirions than virus isolated at the same time from the supernatant medium only; also, the proportion of pseudovirions increased slightly with time after infection. Thus, according to the experimental conditions used, polyoma virus preparations with a low (10 to 20%) or a high (60 to 80%) proportion of pseudovirions can be obtained.

In vitro polyoma DNA synthesis: inhibition by 1-beta-d-arabinofuranosyl CTP

The effects of 1-beta-D-arabinofuranosyl CTP (ara-CTP) on DNA replication were studied in an in vitro system from polyoma-infected BALB/3T3 cells. Ara-CTP concentrations of larger than or equal to 150 muM were found to block in vitro DNA synthesis completely, and concentrations of smaller than or equal to 0.3 muM had no inhibitory effect. Intermediate concentrations resulted in a concentration-dependent reduction of the in vitro synthesis rate. Long-term labeling with [alpha-32-P]ara-CTP demonstrated the incorporation of the analogue into cellular and viral DNA concomitantly with [3-H]TTP. In pulse-labeling experiments, at noninhibitory concentrations of the analogue, ara-CTP was incorporated into short DNA fragments and long growing strands to relatively the same extent as TTP. Partial venom phosphodiesterase digestion liberated the incoporated are-CTP at essentially the same rate as incorporated TTP, excluding a predominantly terminal incorporation, and after total venom phosphodiesterase digestion greater than 80% of the incorporated ara-CTP was recovered as 5'-ara-CMP. Analysis of the long-term in vitro viral DNA product made in the presence of partially inhibiting ara-CTP concentrations demonstrated that none of the steps leading to mature viral DNA were totally inhibited at the ara-CTP concentrations used. Pulse labeling of replicating viral DNA in the presence of ara-CTP revealed two consistent differences in the pattern found in control pulses: (i) predominant labeling of short chains (5S) with reduced amounts of radioactivity in the longer growing viral DNA strands (smaller than or equal to 16S), and (ii) a one-third to one-half reduction in size for short DNA chains labeled in the presence of ara-CTP. Release of the ara-CTP inhibition with excess dCTP resulted in covalent extension of these smaller short chans to approximately the size of regular short chains labeled in the absence of the inhibitor. Isolated short chains synthesized in the presence of ara-CTP exhibited a slightly lower degree of self-complementarity than regular short chains. The predominant labeling of short chains during pulses is, therefore, not a consequence of discontinuous growth on both sides of the replication fork. Similar results were obtained with ara-ATP and N-ethylmaleimide. The experiments indicate that ara-CTP acts primarily on DNA-polymerizing activities, affecting different DNA polymerases to varying degrees. The results are discussed in terms of the possible number and identity of polymerases involved in viral (and cellular) DNA replication.