In vitro polyoma DNA synthesis: Involvement of RNA in discontinuous chain growth

Short fragments of DNA (5 S) isolated by denaturation from polyoma replicative intermediates pulse-labeled in vitro were shown to have RNA covalently attached by three criteria: (1) such fragments were slightly denser than bulk viral DNA. (2) They could be labeled directly with α-³²P-labeled ribotriphosphates. (3) Alkaline hydrolysis of fragments labeled with α-³²P-labeled deoxynucleoside triphosphates showed ³²P transfer to 3′ ribonucleoside monophosphates. Except for a preference of transfer from dC, the link showed little sequence specificity. The data are compatible with the notion that all short fragments in replicating viral DNA are initiated by an RNA primer. This RNA is maximally 30 bases long and is rather short-lived.     

Nitropyrenes are inducers of polyoma viral DNA synthesis

The biological activity of a series of nitropyrenes was assayed by measuring their ability to induce the asynchronous replication of viral DNA in rat fibroblasts transformed by a ts-a mutant of polyoma virus. Concentrations of 10-30 micrograms/ml of 1-nitropyrene (1-NP) induced viral replication, and this effect was enhanced by addition of rat-liver S9 microsomal fraction (300 micrograms/ml) to the culture medium. The response was less than that obtained with 0.1 micrograms/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 replication, in the absence of exogenous microsomal activation, displaying strongly positive effects at 0.5-2.0 microgram/ml. Dose-response curves with 1,6-dinitropyrene (1,6-DNP) from 0.01 to 0.5 microgram/ml indicated 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 modification 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.     

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, 1978a), catalyzes the synthesis of initiator RNA.     

Transcription of polyoma virus DNA in vitro. II. Transcription of superhelical and linear polyoma DNA by RNA polymerase II.

The protein product of the bacteriophage T4 gene 32 is a single-stranded DNA binding protein which functions during phage DNA repair, replication and recombination. Recently the gene 32 protein was shown to participate in the regulation of its own expression. Although the purified protein is known to interact with DNA, the autoregulation was shown to occur at the translational level. The previous analysis in vivo, although coherent, was indirect. We report here direct cell-free experiments in which purified gene 32 protein specifically represses translation of gene 32 messenger RNA.     

Dissection of RNA-primed DNA synthesis catalyzed by gene 4 protein and DNA polymerase of bacteriophage T7. Coupling of RNA primer and DNA synthesis

Gene 4 protein and DNA polymerase of bacteriophage T7 catalyze RNA-primed DNA synthesis on single-stranded DNA templates. T7 DNA polymerase exhibits an affinity for both gene 4 protein and single-stranded DNA, and gene 4 protein binds stably to single-stranded DNA in the presence of dTTP (Nakai, H. and Richardson, C. C. (1986) J. Biol. Chem. 261, 15208-15216). Gene 4 protein-T7 DNA polymerase-template complexes may be formed in both the presence and absence of nucleoside 5'-triphosphates. The protein-template complexes may be isolated free of unbound proteins and nucleotides by gel filtration and will catalyze RNA-primed DNA synthesis in the presence of ATP, CTP, and the four deoxynucleoside 5'-triphosphates. RNA-primed DNA synthesis may be dissected into separate reactions for primer synthesis and DNA synthesis. Upon incubation of gene 4 protein with single-stranded DNA, ATP, and CTP, a primer-template complex is formed; it is likely that gene 4 protein mediates stable binding of the oligonucleotide to the template. The complex, purified free of unbound proteins and nucleotides, supports DNA synthesis upon addition of DNA polymerase and deoxynucleoside 5'-triphosphates. Association of primers with the template is increased by the presence of dTTP or DNA polymerase during primer synthesis. DNA synthesis supported by primer-template complexes initiates predominantly at gene 4 recognition sequences, indicating that primers are bound to the template at these sites.     

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.     

In vitro polyoma DNA synthesis: discontinuous chain growth

Using an in vitro system for polyoma DNA synthesis from polyoma-infected mouse BALB/3T3 cells, we have shown that short pulses of radioactively labeled deoxynucleoside triphosphates are incorporated into viral replicative intermediates. Upon denaturation, the pulse-labeled replicative intermediates yield two size classes of growing DNA chains, namely a heterogeneous long class with S values up to unit viral DNA length (16 S) and a rather discrete short class of 5 S pieces. We have shown that these short fragments are involved as precursors in viral DNA chain elongation and that they can be chased into mature viral DNA. The fragments are found in replicative intermediates at all stages of replication and are therefore presumably not involved in specialized initiation or termination processes. Kinetic analysis of the appearance of radioactivity in short and long chains shows that initially approximately equal amounts are incorporated at a linear rate into the two classes. Subsequently, the rate of incorporation into long chains approximately doubles, while the amount of radioactivity in short chains reaches a plateau. This not only suggests that short chains are precursors to long chains, but that the synthesis of long chains occurs as a separate event and is not simply a result of joining of short fragments. Under the in vitro labeling conditions the time taken for radioactivity in short chains to reach a constant level can be used as a measure of the average lifetime of a 5 S piece. Our analysis indicates that there may be a considerable lag between the completion of a 5 S piece and its joining into longer DNA. Estimates of the self-annealing of the short chains showed approximately 20% self-complementarity. Thus, polyoma synthesis in vitro appears to proceed predominantly by a semi-discontinuous mechanism in which the nascent DNA on one side of the growing fork is elongated continuously, while on the other side of the fork DNA is synthesized discontinuously as 5 S fragments, which are subsequently joined. Both the short and the long chains are synthesized in the 5′ to 3′ direction.A fraction of the pulse-labeled material is found as free 3 to 5 S single-stranded DNA. These pieces are of both viral and cellular origin. A majority of them appear to be involved as precursors in DNA chain elongation.     

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 (Hpall) 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.     

In vitro polyoma DNA synthesis: requirement for cytoplasmic factors.

Purified nuclei from polyoma-infected mouse (3T3) cells were found to be greatly reduced in their ability to synthesize viral DNA in vitro when compared with a crude system consisting of an unfractionated hypotonic lysate of the infected cells. The synthetic capacity of the nuclei could be fully reconstituted when a high-speed cytoplasmic supernatant was added back to them. Cytosols from uninfected mouse, monkey, and hamster cells were equally as effective in stimulating purified nuclei as that of virus-infected mouse cells. Optimal complementation required high concentrations of the cytosol, and most of the complementing activity was destroyed by heating to 60 C. Dialysis had no effect on the activity. Analysis of the viral DNA synthesized in purified nuclei showed an accumulation of Okazaki-type short DNA chains, which could be chased into viral progeny DNA strands if cytosol was added back to the nuclei. Kinetic analysis of the pulse-labeling pattern of viral replicative DNA showed a strong dependence of the extension of viral progeny strands and of the processing of Okazaki-type fragments on the amount of cytosol present during the reaction. It is suggested that the cytoplasmic DNA polymerase might be one of the active components in the cytosol, but most likely not the only one.     

Replication of colicinogenic factor E1 DNA in plasmolysed Escherichia coli cells. Coupling of DNA replication and RNA synthesis.

Plasmolysed chloramphenicol-treated Escherichia coli cells carrying the colicinogenic factor E1 utilize deoxynucleoside triphosphates for the semi-conservative synthesis of Col E1 DNA. Col E1 DNA replication in plasmolysed cells can be dissociated into two temporally separated processes: (a) a rifampicin-sensitive RNA synthesis, which is stimulated by adenosine 3':5'-monophosphate (cyclic AMP) and requires all four ribonucleoside triphosphates and (b) an ATP-dependent DNA synthesis, which is inhibited by arabinosylnucleoside triphosphates and sulfhydryl-blocking reagents. Thes two processes exhibit different sensitivities to inhibition by polyamines and actinomycin D.     

DNA/RNA synthesis and labelling.

Reliable methods of machine-aided RNA synthesis have been established to complement those for DNA assembly. Oligonucleotides containing thio-modified backbones and 2'-O-alkyl sugars head the list of many newly available analogues. Biotin, fluorescent agents and many reporter groups can be conveniently introduced into oligonucleotides in multiples by phosphoramidite or H-phosphonate chemistry.