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: 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.     

Replication of polyoma DNA in isolated nuclei. V. Complementation of in vitro DNA replication.

Nuclei from polyoma-infected 3T6 fibroblasts elongate in vitro the progeny strands of the replicative intermediates of polyoma DNA. When high concentrations of such nuclei were incubated, short DNA fragments were formed and subsequently added onto growing progeny strands. When nuclei were repeatedly washed with buffer containing detergent and then incubated at low concentrations. DNA synthesis was decreased. In particular, the joining process was reduced, resulting in an accumulation of short DNA fragments. All aspects of the synthetic capacity of the nuclei were restored by addition of cytoplasmic extract. Additions of purified enzymes (polynucleotide ligase from calf thymus or Escherichia coli together with E. coli DNA polymerase I) increased the joining function of the nuclei. The system can be used for the identification of the enzymatic steps concerned with polyoma DNA replication.     

Strandedness of newly synthesized short pieces of polyoma DNA from isolated nuclei.

The discontinuous synthesis of the complementary strands of polyoma DNA in isolated nuclei has been studied by hybridization techniques. The relative amounts of the newly synthesized complementary strands were compared by separately annealing them to denatured HpaII restriction fragments. In every case an excess (1.4- to 2.4-fold) of short pieces of the strand growing in the 3' leads to 5' direction was found.     

Both strands of polyoma DNA are replicated discontinuously with ribonucleotide primers in vivo

Nascent polyoma DNA molecules were isolated after pulse-labeling of infected murine 3T6 cells with [3H]thymidine. The extent of digestion of these DNA molecules by spleen exonuclease was increased by exposure to alkali or RNase, suggesting that ribonucleotides were present at or near the 5' terminal of the newly synthesized pieces of DNA. Intermediates shorter than 300 nucleotides were hybridized to the separated strands of restriction enzyme fragments of the polyoma genome: 2.5 to 3-fold more radioactivity was found in the strand whose synthesis is necessarily discontinuous (the lagging strand) than in the strand whose synthesis is potentially continuous (the leading strand) than in the strand whose synthesis is potentially continuous (the leading strand). Separation of the strands of [5'-32P]DNA molecules showed that the excess [3H]thymidine in lagging-strand molecules was not simply the result of an increased number of molecules. Therefore, assuming equivalent efficiencies of labeling, lagging-strand pieces must be slightly longer than those with leading-strand polarity. The presence of ribonucleotides on the 5' termini of molecules with both leading- and lagging-strand polarity was demonstrated by (i) release of 32P-ribonucleoside diphosphates upon alkaline hydrolysis of [5'-32P]DNA separated according to replication polarity and (ii) the change in the degree of self-annealing of nascent molecules upon preferential degradation of DNA molecules possessing initiator RNA moieties by spleen exonuclease. We conclude that replication of polyoma DNA in vivo occurs discontinuously on both sides of the growing fork, using RNA as the major priming mechanism.     

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.     

Hydroxyurea-Induced Accumulation of Short Fragments During Polyoma DNA Replication II. Behavior During Incubation of Isolated Nuclei

The synthesis of polyoma DNA was studied in isolated nuclei from hydroxyurea-inhibited 3T6 cells infected with polyoma virus. During incubation of nuclei under conditions suitable for polyoma DNA synthesis in vitro, the short DNA fragments with a sedimentation coefficient of 4S formed in vivo (hydroxyurea fragments) became associated with preformed, replicating DNA strands. Centrifugation in dye-buoyant density gradients showed that the fragments formed part of the structure of the replicative intermediate of polyoma DNA. The proportion of "young" replicative intermediates was larger after hydroxyurea inhibition than in uninhibited controls. Hydroxyurea fragments appear to be closely related to the 4S fragments formed as normal intermediates during discontinuous synthesis of polyoma DNA.     

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.     

Discontinuous synthesis of both strands at the growing fork during polyoma DNA replication in vitro.

In discontinuous polyoma DNA replication, the synthesis of Okazaki fragments is primed by RNA. During viral DNA synthesis in nuclei isolated from infected cells, 40% of the nascent short DNA fragments had the polarity of the leading strand which, in theory, could have been synthesized by a continuous mechanism. To rule out that the leading strand fragments were generated by degradation of nascent DNA, they were further characterized. DNA fragments from a segment of the genome which replication forks pass in only one direction were strand separated. The sizes of the fragments from both strands were similar, suggesting that one strand was not specifically degraded. Most important, however, the majority of the Okazaki fragments of both strands were linked to RNA at their 5' ends. For identification, the RNA was labeled at the 5' ends by [beta-32P]GTP, internally by [3H]CTP, [3H]GTP, and [3H]UTP, or at the 3' ends by 32P transfer from adjacent [32P]dTMP residues. All three kinds of labeling indicated that an equal proportion of DNA fragments from the two strands was linked to RNA primers.     

Polymerase dynamics at the eukaryotic DNA replication fork

This review discusses recent insights in the roles of DNA polymerases (Pol) delta and epsilon in eukaryotic DNA replication. A growing body of evidence specifies Pol epsilon as the leading strand DNA polymerase and Pol delta as the lagging strand polymerase during undisturbed DNA replication. New evidence supporting this model comes from the use of polymerase mutants that show an asymmetric mutator phenotype for certain mispairs, allowing an unambiguous strand assignment for these enzymes. On the lagging strand, Pol delta corrects errors made by Pol alpha during Okazaki fragment initiation. During Okazaki fragment maturation, the extent of strand displacement synthesis by Pol delta determines whether maturation proceeds by the short or long flap processing pathway. In the more common short flap pathway, Pol delta coordinates with the flap endonuclease FEN1 to degrade initiator RNA, whereas in the long flap pathway, RNA removal is initiated by the Dna2 nuclease/helicase.     

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.     

Adenovirus DNA replication in vitro: Origin and direction of daughter strand synthesis

We have characterized a soluble enzyme system from adenovirus-infected cells that is capable of replicating exogenously added adenovirus DNA in vitro. Maximal DNA synthesis is observed when DNA-protein complex, isolated from purified adenovirus virions, is added as template. Under these conditions DNA replication starts at or near either end of the template. Daughter strand synthesis then proceeds in the 5′ to 3′ direction displacing the parental strand of the same polarity. Thus, the r daughter strand is synthesized from right to left on the conventional map of the adenovirus genome, and the l daughter strand is synthesized from left to right. This course of events is the same as that which occurs during adenovirus DNA replication in vivo. In contrast, when deproteinized adenovirus DNA is added to the in vitro system, the limited DNA synthesis that is observed appears to be due to a repair-like reaction. In particular, synthesis can begin at many sites within the template, and the synthetic product consists largely of short DNA chains that are covalently linked to template DNA strands.     

Replication slippage involves DNA polymerase pausing and dissociation

Genome rearrangements can take place by a process known as replication slippage or copy-choice recombination. The slippage occurs between repeated sequences in both prokaryotes and eukaryotes, and is invoked to explain microsatellite instability, which is related to several human diseases. We analysed the molecular mechanism of slippage between short direct repeats, using in vitro replication of a single-stranded DNA template that mimics the lagging strand synthesis. We show that slippage involves DNA polymerase pausing, which must take place within the direct repeat, and that the pausing polymerase dissociates from the DNA. We also present evidence that, upon polymerase dissociation, only the terminal portion of the newly synthesized strand separates from the template and anneals to another direct repeat. Resumption of DNA replication then completes the slippage process.     

Initiator RNA of nascent DNA from animal cells.

Nascent DNA synthesized by intact cells has been examined for the presence of RNA that may function as a primer in the discontinuous synthesis of DNA. A low molecular weight fraction that contains nascent DNA was isolated from a human lymphoblastoid cell line in logarithmic growth. After labeling the 5′ ends with bacteriophage T4 polynucleotide kinase and [γ-³²P]ATP, and digestion of the DNA with DNAase, a DNAase-resistant oligonucleotide was isolated. This fragment consisted of approximately 9 ribonucleotide residues, with 5′ terminal purines ($\text{A}\text{G}\text{=}\text{3·5}\text{1}$), plus one to three 3′ terminal deoxynucleotides resulting from incomplete removal by DNAase. Approximately 10% of short nascent DNA chains contained the nonanucleotide molecule. An additional 20% of the nascent DNA contained ribooligomers shorter than 9 residues, with 5′ termini substantially increased in pyrimidines, which may result from degradation of the nonanucleotide. These results extend previous studies that demonstrated a similar ribooligonucleotide present at the 5′ end of most or all short nascent DNA chains synthesized in broken cell systems. Together with the results obtained by Reichard and co-workers (Reichard et al., 1974) with polyoma virus, the data support a mechanism by which a short initiator RNA serves as primer for discontinuously synthesized DNA in animal cells.     

Highly efficient DNA synthesis by the phage phi 29 DNA polymerase. Symmetrical mode of DNA replication

The results presented in this paper indicate that the phi 29 DNA polymerase is the only enzyme required for efficient synthesis of full length phi 29 DNA with the phi 29 terminal protein, the initiation primer, as the only additional protein requirement. Analysis of phi 29 DNA polymerase activity in various in vitro DNA replication systems indicates that two main reasons are responsible for the efficiency of this minimal system: 1) the phi 29 DNA polymerase is highly processive in the absence of any accessory protein; 2) the polymerase itself is able to produce strand displacement coupled to the polymerization process. Using primed M13 DNA as template, the phi 29 DNA polymerase is able to synthesize DNA chains greater than 70 kilobase pairs. Furthermore, conditions that increase the stability of secondary structure in the template do not affect the processivity and strand displacement ability of the enzyme. Thus, the catalytic properties of the phi 29 DNA polymerase are appropriate for a phi 29 DNA replication mechanism involving two replication origins, strand displacement and continuous synthesis of both strands. The enzymology of phi 29 DNA replication would support a symmetrical model of DNA replication.     

A cloned polyoma DNA fragment representing the 5'' half of the early gene region is oncogenic.

The two polyoma DNA fragments generated by cleavage with BamHI and EcoRI were cloned in pBR322, and their oncogenic potential was tested in vivo and in vitro. Only recombinant plasmid DNA containing a polyoma DNA fragment which extends clockwise from 58 to 0 map units and include approximately the 5'-proximal half of the early gene region produced tumors in newborn hamsters and transformed rat embryo cells in tissue culture. Southern blotting analysis indicated that the entire 2.2-kilobase polyoma BamHI-EcoRI fragment was intact in both a tumor cell line and a cell line transformed in culture which we examined. The presence of polyoma middle and small T antigen in these lines was demonstrated by immunoprecipitation and tryptic peptide mapping. DNA from a recombinant plasmid containing a polyoma genome deleted between 90 and 4 map units failed to induce tumors or transform cells.     

Regulation of DNA chain elongation in SV3T3 cells in relation to growth rate.

Regulation of DNA synthesis was investigated in SV40 transformed 3T3 cells exhibiting variable growth rates and residence times in S phase when cultured in the presence of different serum concentrations. Pulse-labeled DNA was chased into large molecular weight material in vivo much more slowly in slowly growing cells than in cells growing at the normal rate. Consistent with this, the joining of short (less than 10 S) chains to form long (greater than 10 S) chains by whole cell lysate system in vitro was greatly impaired in slowly growing cells compared to controls. Thus the lengthening of S phase in SV3T3 cells growing slowly in low serum is reflected in a reduced rate of DNA chain elongation. The presence of cycloheximide during chase in vivo reduced the rate of conversion of pulse-labeled molecules into large molecular weight DNA in both slowly growing and normally growing cells.