Replication of polyoma DNA in isolated nuclei. 3. The nucleotide sequence at the RNA-DNA junction of nascent strands

Short fragments consisting of about 100 to 140 deoxyribonucleotides serve as intermediates in the elongation of polyoma DNA. In nuclei isolated from polyoma-infected 3T6 mouse fibroblasts these fragments are initiated by stretches of RNA. We investigated the nature of the ribo- and deoxyribonucleotides at the RNA-DNA link. DNA was synthesized in vitro from each of the four α-³²P-labelled deoxynucleoside triphosphates, the nascent strands were hydrolysed with alkali and the transfer of isotope to ribonucleotides was studied after fractionation of strands according to size. Each strand contained on the average one RNA-DNA link at the 5′ end of DNA. All four common ribo- and deoxyribonucleotides were present at the RNA-DNA link with close to equal frequency, irrespective of chain length or incubation time.In a second approach, daughter strands synthesized in vivo were treated with alkali and the 5′-OH ends of DNA liberated were ³²P-labelled using polynucleotide kinase. All four deoxynucleotides were labelled by this treatment confirming the corresponding results of the in vitro experiments.During the discontinuous synthesis of polyoma DNA the switch from RNA to DNA synthesis is thus not effected by a specific sequence at the RNA-DNA junction, in contrast to Escherichia coli where the sequence p(rPy)p(dC)p was reported.     

Biosynthetic studies of the Y base in yeast phenylalanine tRNA. Incorporation of guanine

Replicating polyoma virus DNA, pulse-labeled with ³H-thymidine, was isolated from infected mouse embryo cells by velocity sedimentation in neutral sucrose and purified by benzoylated-naphthoylated DEAE-cellulose chromatography. Nascent strands, prepared by heat denaturation of purified replicative intermediate, banded at a slightly higher buoyant density in neutral cesium sulfate gradients than single strands derived from superhelical viral DNA. Treatment of nascent strands with a mixture of ribonucleases 1A and T1 shifted their buoyant density to that of single strands derived from superhelical viral DNA. These results indicate that an oligoribonucleotide component is covalently associated with replicating polyoma DNA strands.     

An electron microscopic method for studying and mapping the region of weak sequence homology between simian virus 40 and polyoma DNAs.

A procedure for investigating the possibility of small amounts of partial DNA sequence homology between two defined DNA molecules has been developed and used to test for sequence homology between simian virus 40 and polyoma DNAs. This procedure, which does not necessitate the use of separated viral DNA strands, involves the construction of hybrid DNA molecules containing a simian virus 40 DNA molecule covalently joined to a polyoma DNA molecule, using the sequential action of EcoRI restriction endonuclease and Escherichia coli DNA ligase. Denaturation of such hybrid DNA molecules then makes it possible to examine intramolecularly rather than intermolecularly renatured molecules. Visualization of these intramolecularly renatured “snapback” molecules with duplex regions of homology by electron microscopy reveals a 15% region of weak sequence homology. This region is denatured at about 35 °C below the melting temperature of simian virus 40 DNA and therefore corresponds to about 75% homology. This region was mapped on both the simian virus 40 and polyoma genomes by the use of Hemophilus parainfluenzae II restriction endonuclease cleavage of the simian virus 40 DNA prior to EcoRI cleavage and construction of the hybrid molecule. The 15% region of weak homology maps immediately to the left of the EcoRI restriction endonuclease cleavage site in the simian virus 40 genome and halfway around from the EcoRI restriction endonuclease cleavage site in the polyoma genome.     

Stimulation of polyoma DNA replication in isolated nucleoprotein complexes by factors from Drosophila embryos

Nucleoprotein complexes containing both form 1 and replicative intermediates of polyoma DNA prepared from nuclei of virus-infected mouse fibroblasts retain a limited ability to elongate progeny strands of the replicative intermediates. Compared to isolated nuclei, both the rate and the extent of strand elongation is greatly decreased. The isolated complexes synthesize initiator RNA and start new Okazaki fragments, but are deficient in the joining of these fragments. Addition of small amounts of an extract from 16 hours old Drosophila embryos corrects the deficiencies. The stimulatory activity of the extract can be partially purified and has been separated into two fractions by chromatography on Sepharose 6B. With immunological techniques we demonstrate that the mouse DNA polymerase-α, tightly bound to the complexes, is responsible for DNA strand elongation.The Drosophila α-polymerase present in one of the two fractions purified on Sepharose 6B cannot substitute for the mouse enzyme. The stimulatory activity of the Drosophila fractions is thus not due to α-polymerase.     

Transcription of polyoma virus DNA in vitro. III. Localization of calf thymus RNA polymerase II binding sites.

The binding sites of calf thymus RNA polymerase II on polyoma DNA were monitored by electron microscopy. Six discrete binding sites were located at positions 0.06, 0.25, 0.57, 0.66, 0.85 and 0.98 on the physical map of polyoma DNA. Although most of these sites are located in easily denaturable regions of the DNA, the strongest binding sites do not overlap with the major A + T-rich regions. In addition, the same binding sites were observed on superhelical or linear polyoma DNA. These results suggest that the eucaryotic RNA polymerase II can recognize specific sequences on double-stranded DNA and not only easily denaturable regions. At least five of these sites correspond to the binding and initiation sites mapped previously for the Escherichia coli RNA polymerase (Lescure et al., 1976).Stable initiation complexes can be formed with both E. coli and calf thymus RNA polymerases in the presence of a single dinucleotide (GpU) and a specific ribotriphosphate (CTP). Under these conditions, the binding of both enzymes to the sites in positions 0.06 and 0.57 is stimulated whereas the binding in positions 0.65 and 0.84 is partially suppressed. Both eucaryotic and procaryotic RNA polymerases may recognize similar sequences of the viral DNA in vitro.     

Covalent attachment of RNA to nascent DNA in mammalian cells

We have used the technique of phosphate transfer analysis to test for the presence of phosphodiester bonds linking ribonucleotides (on the 5′ side) to deoxyribonucleotides (on the 3′ side) in DNA newly synthesized within lysates or purified nuclei of mammalian cells. We have found that such covalent junctions between RNA and DNA are present at a frequency of one junction per newly synthesized DNA strand. The junctions are located close to the ends of the nascent DNA strands. The stretches of RNA at the junction are very short compared to the stretches of DNA. These properties are consistent with the conclusion by Reichard, Eliasson, and Söderman (1974) that short stretches of RNA are present on the 5′ ends of nascent DNA strands produced during replication of polyoma virus.     

DNA synthesis in polyoma virus infection. II. Relationship between viral DNA replication and initiation of cellular DNA replicons

The rate of synthesis of cellular DNA is stimulated in stationary phase mouse embryo cells infected with polyoma virus. Nascent cellular DNA strands pulselabeled with [³H]thymidine in the presence of replicating viral DNA are smaller, by an average of 2·1 × 10⁷ daltons, than DNA made under similar conditions in uninfected cells. Previous work (Cheevers et al., 1972) has indicated that this observation is the consequence of activation in infected cells of cellular DNA initiation sites not in operation during a similar pulse-labeling interval in uninfected cells. Similar results were obtained using cells infected with the temperature-sensitive Ts-a mutant of polyoma at 32 °C, which permits both the induction of cellular DNA synthesis and replication of viral DNA. However, at a temperature of 39 °C, which permits only the induction of cellular DNA replication in Ts-a-infected cells, the size of newly synthesized DNA is not different from that of uninfected cells. Similarly, in rat embryo cells abortively infected with polyoma (wild-type), stimulation of cellular DNA synthesis occurs but viral DNA replication is restricted, and no difference is apparent in the size of newly formed DNA as compared to uninfected cells. These results are interpreted to mean that in productively infected cells, polyoma DNA and some regions of the host genome may be co-ordinately replicated.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication I. Characterization of Fragments

Hydroxyurea treatment of 3T6 mouse fibroblast cells infected with polyoma virus resulted within 15 min in more than a 20-fold reduction of the rate of both viral and cellular DNA synthesis. After the initial rapid inhibition, the rate of DNA synthesis remained essentially constant for at least 2 h. In the inhibited cells viral DNA accumulated as short chains with a sedimentation coefficient of about 4S (hydroxyurea fragments). A variable proportion of these fragments was released from the template strands when the viral DNA was extracted by the Hirt procedure. Reannealing experiments demonstrated that hydroxyurea fragments were polyoma-specific and probably synthesized on both parental strands at the replication forks.     

Structural Studies of Heterochiral DNA/DNA,RNA/RNA,AND DNA/RNA Duplexes

Using DNA and RNA heptanucleotides containing an unnatural L-nucleotides as well as the complementary strands, effects of the introduction of an L-nucleotide on the structure of DNA/DNA, RNA/RNA, and DNA/RNA duplexes were investigated by circular dichroism experiments and RNase H-mediated RNA strand cleavage reaction. The results suggested that the substitution of the central D-nucleotide with an L-nucleotide in the duplexes causes the significant structural alterations as the duplex structures change to conformations with more B-form similarities.     

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.     

Synthesis and repair of RNA-containing supercoiled plasmid ColE1 DNA in Escherichia coli

Supercoiled plasmid molecules sensitive to nicking by RNase or alkali have been shown to accumulate during replication of colicinogenic factor E1 (ColE1) in Escherichia coli in the presence of chloramphenicol. The possibility that this sensitivity is due to the covalent integration of RNA molecules during the synthesis of plasmid DNA is supported by the demonstration that (a) strands of supercoiled ColE1 newly replicated in the presence of chloramphenicol exhibit sensitivity to RNase and alkali treatment, while (b) RNase- and alkali-resistant circular strands of plasmid DNA synthesized either before or after the addition of chloramphenicol remain resistant during subsequent replication of the plasmid in the presence of chloramphenicol. Furthermore, newly made plasmid DNA strands cannot act as templates for further rounds of replication if they possess an RNA segment. The existence of a repair mechanism for the removal of the RNA segment from supercoiled ColE1 DNA molecules was demonstrated by pulse-chase experiments. It was observed that the proportion of RNase-sensitive molecules is considerably higher in pulse-labeled as compared to continuously labeled ColE1 DNA synthesized in the presence of chloramphenicol, and the proportion of pulse-labeled ColE1 DNA that is RNase sensitive is greatly reduced during a chase period. Removal of the RNA segment is also carried out effectively at the restrictive temperature in temperature-sensitive DNA polymerase I mutants. In a survey of other bacterial mutants defective in the repair of damaged DNA, a substantial increase in the rate of accumulation of RNase-and alkali-sensitive supercoiled ColE1 DNA in the presence of chloramphenicol was observed in recBC and uvrA mutants in comparison with the wild-type strains.