RNA primers in Simian virus 40 DNA replication. II. Distribution of 5' terminal oligoribonucleotides in nascent DNA.

A cell-free simian virus 40 (SV40) DNA replication system served to study the role of RNA in the initiation of nascent DNA chains of less than 200 nucleotides (Okazaki pieces). RNA-DNA covalent linkages were found to copurify with SV40 replicating DNA. These linkages were identified by transfer of a fraction of the ³²P from the 5′ position of a deoxyribonucleotide to 2′(3′)rNMPs upon either alkaline hydrolysis or RNAase T₂ digestion of SV40 replicating [³²P]DNA. Alkaline hydrolysis also exposed 5′ terminal hydroxyl groups in the nascent DNA which were detected as nucleosides after digestion with P1 nuclease. The RNA-DNA covalent linkages resulted from a population of Okazaki pieces containing uniquely sized oligoribonucleotides covalently attached to their 5′ termini (RNA primers). The density of a portion of the Okazaki pieces in potassium iodide gradients corresponded to a content of 90% DNA and 10% RNA, while the remaining Okazaki pieces appeared to contain only DNA. Incubation of Okazaki pieces with a defined length in the presence of either RNAase T₂ or potassium hydroxide converted about one-third to one-half of them intto a second well defined group of DNA chains of greater electrophoretic mobili y in polyacrylamide gels. The increased mobility corresponded to the removalof at least seven-residues. Since alkaline hydrolysis of similar Okazaki pieces revealed that one-third to one-half of them contained rN-³²P-dN linkages, the oligoribonucleotides must be covalently attached to the 5′ ends of nascent DNA chains. Although the significance of two populations of Okazaki pieces, one with and one without RNA primers, is imperfectly understood, a sizable fraction of nascent DNA chains clearly contained RNA primers.Neither the length of the RNA primer nor the number of RNA primers per DNA chain changed significantly with increasing length of Okazaki pieces. Since the frequency of RNA-DNA junctions found in nascent DNA chains greater than 400 nucleotides was similar to that of Okazaki pieces, the complete excision of RNA primers appears to occur after Okazaki pieces are joined to the 5′ end of growing daughter strands.³²P-label transfer analysis of Okazaki pieces recovered from hybrids with isolated HindII + III restriction fragments of SV40 DNA revealed a uniform distribution of rN-P-dN sequences around the replicating DNA molecule. Therefore, most, if not all, RNA primers serve to initiate Okazaki pieces rather than to initiate DNA replication at the origin of the genome. Moreover, the positions of RNA primers are not determined by a specific set of nucleotide sequences.     

Assembly of simian virus 40 Okazaki pieces from DNA primers is reversibly arrested by ATP depletion.

We have previously proposed that DNA polymerase alpha-primase provides short RNA-DNA precursors below 40 nucleotides (DNA primers), several of which assemble into an Okazaki piece after intervening RNA has been removed and the gaps have been filled by DNA polymerase delta (or epsilon) (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988; T. Nethanel and G. Kaufmann, J. Virol. 64:5912-5918, 1990). In this report, we confirm and extend these conclusions by studying the effects of deoxynucleoside triphosphate (dNTP) concentrations and the presence of ATP on the occurrence, dynamics, and configuration of DNA primers in simian virus 40 replicative intermediate DNA. We first show that these parameters are not significantly affected by a 10-fold increase in dNTP precursor concentrations. We then demonstrate that Okazaki piece synthesis can be arrested at the level of DNA primers by ATP depletion. The arrested DNA primers faced short gaps of 10 to 20 nucleotides at their 3' ends and were progressively chased into Okazaki pieces when ATP was restored. ATP could not be substituted in this process by adenosine-5'-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. The chase was interrupted by aphidicolin but not by butylphenyl-dGTP. The results implicate an ATP-requiring factor in the switch between the two DNA polymerases engaged in Okazaki piece synthesis. They also suggest that the replication fork advances by small, DNA primer-size increments.     

Metabolism of Okazaki fragments during simian virus 40 DNA replication.

Essentially all of the Okazaki fragments on replicating Simian virus 40 (SV40)DNA could be grouped into one of three classes. Class I Okazaki fragments (about 20%) were separated from longer nascent DNA chains by a single phosphodiester bond interruption (nick) and were quantitatively identified by treating purified replicating DNA with Escherichia coli DNA ligase and then measuring the fraction of Okazaki fragments joined to longer nascent DNA chains. Similarly, class II Okazaki fragments (about 30%) were separated by a region of single-stranded DNA template (gap) that could be filled and sealed by T4 DNA polymerase plus E. coli DNA ligase, and class III fragments (about 50%) were separated by RNA primers that could be removed with E. coli DNA olymerase I, allowing the fragments to be joined with E. coli DNA ligase. These results were obtained with replicating SV40 DNA that had been briefly labeled with radioactive precursors in either intact cells or isolated nuclei. When isolated nuclei were further incubated in the presence of cytosol, all of the Okazaki fragments were converted into longer DNA strands as expected for intermediates in DNA synthesis. However, when washed nuclei were incubated in the abscence of cytosol, both class I and class II Okazaki fragments accumulated despite the excision of RNA primers: class III Okazaki fragments and RNA-DNA covalent linkages both disappeared at similar rates. These data demonstrate the existence of RNA primers in whole cells as well as in isolated nuclei, and identify a unique gap-filling step that is not simply an extension of the DNA chain elongation process concomitant with the excision of RNA primers. One or more factos found in cytosol, in addition to DNA polymerase alpha, are specifically involved in the gap-filling and ligation steps. The sizes of mature Okazaki fragments (class I) and Okazaki fragments whose synthesis was completed by T4 DNA polymerase were measured by gel electrophoresis and found to be broadly distributed between 40 and 290 nucleotides with an average length of 135 nucleotides. Since 80% and 90% of the Okazaments does not occur at uniformly spaced intervals along the DNA template. During the excision of RNA primers, nascent DNA chains with a single ribonucleotide covalently attached to the 5' terminus were identified as transient intermediates. These intermediates accumulated during excision of RNA primers in the presence of adenine 9-beta-D-arabinoside 5'-triphosphate, and those Okazaki fragments blocked by RNA primers (class III) were found to have originated the farthest from the 5' ends of long nascent DNA strands. Thus, RNA primers appear to be excised in two steps with the second step, removal of the final ribonucleotide, being stimulated by concomitant DNA synthesis. These and other data were used to construct a comprehensive metabolic pathway for the initiation, elongation, and maturation of Okazaki fragments at mammalian DNA replication forks.     

Two DNA polymerases may be required for synthesis of the lagging DNA strand of simian virus 40

Agents discriminating between DNA polymerase alpha and DNA polymerases of class delta (polymerase delta or epsilon) were used to characterize steps in the synthesis of the lagging DNA strand of simian virus 40 during DNA replication in isolated nuclei. The synthesis of lagging-strand intermediates below 40 nucleotides, termed DNA primers (T. Nethanel, S. Reisfeld, G. Dinter-Gottlieb, and G. Kaufmann, J. Virol. 62:2867-2873, 1988), was selectively inhibited by butylphenyl dGTP or by neutralizing DNA polymerase alpha monoclonal antibodies. The synthesis of longer lagging chains of up to 250 nucleotides (Okazaki pieces) was affected to a lesser extent, possibly indirectly, by these agents. Aphidicolin, which inhibits both alpha- and delta-class enzymes, elicited the opposite pattern: DNA primers accumulated in its presence and were not converted into Okazaki pieces. These and previous data suggest that DNA polymerase alpha primase synthesizes DNA primers, whereas another DNA polymerase, presumably DNA polymerase delta or epsilon, mediates the conversion of DNA primers into Okazaki pieces.     

Asymmetric Okazaki piece synthesis during replication of simian virus 40 DNA in vivo.

We have pulse-labeled simian virus 40 (SV40)-infected monkey cells with ³H-thymidine (³H-dThd) and have hybridized the viral Okazaki pieces (rapidly labeled short DNA chains found during DNA replication, < 250 nucleotides long) and SV40 “intermediate sized” DNA (longer nascent strands, up to full replicon size) to the separated strands of two SV40 DNA restriction fragments, one lying to either side of the origin of bidirectional DNA replication. As much as 5 fold more Okazaki piece DNA hybridized to one strand than to the other strand of each restriction fragment. The excess Okazaki piece DNA was in the strands oriented 3′ → 5′ away from the replication origin (the strands which are expected to be synthesized discontinuously). Neither the duration of the labeling period nor the temperature of the cells during labeling significantly altered this hybridization asymmetry. With respect to the hybridization of “intermediate sized” DNA, a reverse asymmetry was detected (1.7 fold more radioactivity in the strands oriented 5′ → 3′ away from the origin for a 1 min pulse label at 22°C). The effects on these hybridization asymmetries of preincubating the infected cells with FdUrd prior to pulse-labeling were also determined.We also measured the size of the Okazaki pieces using gel electrophoresis under denaturing conditons after releasing the pieces from the filter-bound DNA strands. The size distribution of the Okazaki piece DNA from each strand was the same (～ 145 nucleotides, weight average; 200–250 nucleotides, maximum size), indicating that the hybridization asymmetry resulted from a difference in the number rather than the size of the pieces in each strand.The simplest interpretation of our results is that SV40 DNA is synthesized semidiscontinuously: the strand with 3′ → 5′ orientation away from the origin is synthesized in short Okazaki pieces which are subsequently joined together, while the strand with 5′ → 3′ orientation away from the origin is synthesized continuously. Some models of two-strand discontinuous synthesis, however, cannot be ruled out.     

Uncoupling of SV40 tsA replicon activation from DNA chain elongation by temperature shifts and aphidicolin arrest.

To synchronize SV40 replicons, simian cells infected with a tsA mutant were restricted at 40 degrees, to complete ongoing replication and returned to 32 degrees, to activate new replicons in the presence of the DNA chain elongation inhibitor aphidicolin. Upon further incubation at 40 degrees without the drug, 3H-dT was incorporated into SV40 FI DNA, almost to the extent seen with cells recovered in the absence of the drug. To determine whether DNA synthesis would begin from the origin, following the temperature-shifts-aphidicolin regimen, chains subsequently pulse-labeled with (alpha-32p)dGTP in isolated nuclei were analyzed for size distribution and genomic location. These chains reached up to 300-400 nucleotides in size, unlike the control which featured comparable amounts of label in long chains and Okazaki pieces. The nascent DNA of the drug-treated system could be chased into longer chains, indicating that it was a replicative intermediate; and it hybridized preferentially to an origin proximal fragment of AtuI- restricted SV40 DNA, demonstrating partial replicon synchronization. The data prove that T-antigen activates the SV40 replicon independent of DNA chain elongation and suggest means to study the mechanism of DNA chain priming at the origin.     

Selection of template initiation sites and the lengths of RNA primers synthesized by DNA primase are strongly affected by its organization in a multiprotein DNA polymerase alpha complex.

Synthesis of (p)ppRNA-DNA chains by purified HeLa cell DNA primase-DNA polymerase alpha (pol alpha-primase) was compared with those synthesized by a multiprotein form of DNA polymerase alpha (pol alpha 2) using unique single-stranded DNA templates containing the origin of replication for simian virus 40 (SV40) DNA. The nucleotide locations of 33 initiation sites were identified by mapping G*pppN-RNA-DNA chains and identifying their 5'-terminal ribonucleotide. Pol alpha 2 strongly preferred initiation sites that began with ATP rather than GTP, thus frequently preferring different initiation sites than pol alpha-primase, depending on the template examined. The initiation sites selected in vitro, however, did not correspond to the sites used during SV40 DNA replication in vivo. Pol alpha 2 had the greatest effect on RNA primer size, typically synthesizing primers 1-5 nucleotides long, while pol alpha-primase synthesized primers 6-8 nucleotides long. These differences were observed even at individual initiation sites. Thus, the multiprotein form of DNA primase-DNA polymerase alpha affects selection of initiation sites, the frequency at which the sites are chosen, and length of RNA primers.     

The replication of DNA containing the simian virus 40 origin by the monopolymerase and dipolymerase systems.

The influence of DNA polymerase (pol) alpha and DNA primase on SV40 DNA replication was examined in both the monopolymerase and dipolymerase systems. The synthesis of oligoribonucleotides in the monopolymerase and dipolymerase systems, followed by pulse labeling with deoxynucleoside triphosphates, yielded short Okazaki fragments approximately 35 nucleotides in length that were chased into full-length Okazaki fragments with time. In the presence of activator 1 and proliferating cell nuclear antigen (PCNA), but no pol delta, these short fragments hardly increased in size with time. DNA fragments of similar size (approximately 35 nucleotides) were previously observed in SV40 replication reactions carried out with crude extracts of HeLa cells in the presence of antibodies directed against PCNA (Bullock, P. A., Seo, Y.S., and Hurwitz, J. (1991) Mol. Cell. Biol. 11, 2350-2361). Thus, the pol alpha-primase complex appears to act processively for only a short distance. At high levels of pol alpha and primase, both short and long DNA products were formed in both systems. In the presence of limiting amounts of pol alpha and excess primase, the monopolymerase system inefficiently yielded longer length Okazaki fragments than those formed with excess pol alpha and primase, whereas the dipolymerase system yielded both short and long DNA fragments. In the presence of limiting amounts of primase and excess pol alpha, long products were formed in both systems, and virtually no short products accumulated. Thus, the ratio between the polymerase and primer ends available controls the size of the nascent product DNA strands. We examined whether PCNA, the T4 phage-encoded gene product 45 (T4 gp45), and the Escherichia coli beta subunit of DNA polymerase III (dnaN gene product) supported SV40 DNA replication and the elongation of single-stranded DNA-binding protein-coated singly primed DNA in reactions catalyzed by pol delta, T4 DNA pol, and E. coli DNA pol III*, respectively. In the presence of T4 gp44/62 and T4 gp32 (but not human single-stranded DNA-binding protein isolated from HeLa cells), T4 DNA pol was weakly activated by PCNA and the beta subunit in lieu of T4 gp45 in the elongation of singly primed phi X174 DNA. However, the other systems were specific for their analogous auxiliary factors. This specificity indicates the importance of protein-protein interactions.     

An oligoribonucleotide polymerase from SV40-infected cells with properties of a primase

A transient decaribonucleotide (iRNA) is covalently linked to nascent eukaryotic DNA chains at their 5' end. Searching for the putative iRNA polymerase (primase), we detected in extracts from SV40-infected cells a DNA-dependent incorporation of UMP residues from UTP into free and DNA linked deca- or similarly sized ribonucleotides. Denatured salmon sperm DNA served as the standard template in this reaction. SV40 FIII DNA was also an effective template, SV40 FII DNA was ineffective while FI yielded mainly free decaribonucleotides. The incorporation depended on the other rNTPs and was resistant to high concentrations of alpha-amanitin and rifamycin AF/013, drugs inhibitory to RNA polymerases I, II and III. The results implicate the decaribonucleotide polymerase in the priming of nascent DNA chains and suggest that the unique size of iRNA is encoded within its primase.     

The role of RNA primer in discontinuous DNA replication in isolated nuclei from HeLa cells

RNA-primed discontinuous DNA synthesis was studied in an in vitro system consisting of washed nuclei from synchronized S-phase HeLa cells. A new technique proved useful for the purification of short nascent fragments of DNA (Okazaki fragments). Mercurated dCTP was substituted for dCTP in the DNA synthesis reaction. Short nascent pieces (4–6 S) of mercurated DNA were found to bind preferentially to sulfhydryl-agarose, and could be eluted with mercaptoethanol. The isolated fragments were assayed for the presence of covalently linked RNA by the spleen exonuclease method described by Kurosawa et al. (Kurosawa, Y., Ogawa, T., Hirose, S., Okazaki, T. and Okazaki, R. (1975) J. Mol. Biol. 96, 653–664). Following a 30 s incubation with [³H]TTP in the absence of added ribonucleotides, approximately 20% of the nascent strands synthesized in washed nuclear preparations had RNA attached. These RNA primers either preexisted in the nuclei or were formed from endogenous ribonucleotides. The 5′ ends of the primers appeared to be largely in a phosphorylated state. In the absence of added ribonucleotides, these RNA-DNA linkages disappeared within 2 min, whereas if ribonucleotides were added, the number of RNA primers increased to 40% and remained at this level for greater than 2 min. To obtain maximal levels of RNA primer, the addition of all three of the ribonucleotides, rCTP, rGTP and rUTP (0.1 mM), as well as high levels of rATP (5 mM) was required. Addition of ribonucleotides also markedly enhanced the amount of nascent DNA fragments synthesized. However, in the absence of added ribonucleotides, after RNA primers had disappeared, nascent DNA fragments were still initiated at a significant rate. These results suggest that RNA primers play an important role in the initiation of Okazaki fragments but that synthesis can also be initiated by alternative mechanisms. An important role for ATP in RNA primer synthesis is suggested.     

Initiation of simian virus 40 DNA replication in vitro: aphidicolin causes accumulation of early-replicating intermediates and allows determination of the initial direction of DNA synthesis.

Aphidicolin, a specific inhibitor of DNA polymerase alpha, provided a novel method for distinguishing between initiation of DNA synthesis at the simian virus 40 (SV40) origin of replication (ori) and continuation of replication beyond ori. In the presence of sufficient aphidicolin to inhibit total DNA synthesis by 50%, initiation of DNA replication in SV40 chromosomes or ori-containing plasmids continued in vitro, whereas DNA synthesis in the bulk of SV40 replicative intermediate DNA (RI) that had initiated replication in vivo was rapidly inhibited. This resulted in accumulation of early RI in which most nascent DNA was localized within a 600- to 700-base-pair region centered at ori. Accumulation of early RI was observed only under conditions that permitted initiation of SV40 ori-dependent, T-antigen-dependent DNA replication and only when aphidicolin was added to the in vitro system. Increasing aphidicolin concentrations revealed that DNA synthesis in the ori region was not completely resistant to aphidicolin but simply less sensitive than DNA synthesis at forks that were farther away. Since DNA synthesized in the presence of aphidicolin was concentrated in the 300 base pairs on the early gene side of ori, we conclude that the initial direction of DNA synthesis was the same as that of early mRNA synthesis, consistent with the model proposed by Hay and DePamphilis (Cell 28:767-779, 1982). The data were also consistent with initiation of the first DNA chains in ori by CV-1 cell DNA primase-DNA polymerase alpha. Synthesis of pppA/G(pN)6-8(pdN)21-23 chains on a single-stranded DNA template by a purified preparation of this enzyme was completely resistant to aphidicolin, and further incorporation of deoxynucleotide monophosphates was inhibited. Therefore, in the presence of aphidicolin, this enzyme could initiate RNA-primed DNA synthesis at ori first in the early gene direction and then in the late gene direction, but could not continue DNA synthesis for an extended distance.     

Simian Virus 40 DNA Replication in Isolated Replicating Viral Chromosomes

Three subnuclear systems capable of continuing many aspects of simian virus 40 (SV40) DNA replication were characterized in an effort to define the minimum requirements for "normal" DNA replication in vitro. Nuclear extracts, prepared by incubating nuclei isolated from SV40-infected CV-1 cells in a hypotonic buffer to release both SV40 replicating and mature chromosomes, were either centrifuged to separate the total SV40 nucleoprotein complexes from the soluble nucleosol or fractionated on sucrose gradients to provide purified SV40 replicating chromosomes. With nuclear extracts, CV-1 cell cytosol stimulated total DNA synthesis, elongation of nascent DNA chains, maturation and joining of "Okazaki pieces," and the conversion of replicating viral DNA into covalently closed, superhelical DNA. Nucleoprotein complexes responded similarly, but frequently the response was reduced by 10 to 30%. In contrast, isolated replicating chromosomes in the presence of cytosol appeared only to complete and join Okazaki pieces already present on the template; without cytosol, Okazaki pieces incorporated alpha-(32)P-labeled deoxynucleoside triphosphates but failed to join. Consequently, replicating chromosomes failed to extensively continue nascent DNA chain growth, and the conversion of viral replicating DNA into mature DNA was seven to eight times less than that observed in nuclear extracts. Addition of neither cytosol nor nucleosol corrected this problem. In the presence of cytosol, nonspecific endonuclease activity was not a problem in any of the three in vitro systems. Extensive purification of replicating chromosomes was limited by three as yet irreversible phenomena. First, replicating chromosomes isolated in a low-ionic-strength medium had a limited capability to continue DNA synthesis. Second, diluting either nuclear extracts or replicating chromosomes before incubation in vitro stimulated total DNA synthesis but was accompanied by the simultaneous appearance of small-molecular-weight nascent DNA not associated with intact viral DNA templates and a decrease in the synthesis of covalently closed viral DNA. Although this second phenomenon appeared similar to the first, template concentration alone could not account for the failure of purified replicating chromosomes to yield covalently closed DNA. Finally, preparation of nucleoprotein complexes in increasing concentrations of NaCl progressively decreased their ability to continue DNA replication. Exposure to 0.3 M NaCl removed one or more factors required for DNA synthesis which could be replaced by addition of cytosol. However, higher NaCl concentrations yielded nucleoprotein complexes that had relatively no endogenous DNA synthesis activity and that no longer responded to cytosol. These data demonstrate that continuation of endogenous DNA replication in vitro requires both the soluble cytosol fraction and a complex nucleoprotein template whose ability to continue DNA synthesis depends on its concentration and ionic environment during its preparation.     

Structure of chromatin at deoxyribonucleic acid replication forks: location of the first nucleosomes on newly synthesized simian virus 40 deoxyribonucleic acid

Exonucleases specific for either 3' ends (Escherichia coli exonuclease III) or 5' ends (bacteriophage T7 gene 6 exonuclease) of nascent DNA chains have been used to determine the number of nucleotides from the actual sites of DNA synthesis to the first nucleosome on each arm of replication forks in simian virus 40 (SV40) chromosomes labeled with [3H]thymidine in whole cells. Whereas each enzyme excised all of the nascent [3H]DNA from purified replicating SV40 DNA, only a fraction of the [3H]DNA was excised from purified replicating SV40 chromosomes. The latter result was attributable to the inability of either exonuclease to digest nucleosomal DNA in native replicating SV40 chromosomes, as demonstrated by the following observations: (i) digestion with either exonuclease did not reduce the amount of newly synthesized nucleosomal DNA released by micrococcal nuclease during a subsequent digestion period; (ii) in briefly labeled molecules, as much as 40% of the [3H]DNA was excised from long nascent DNA chains; (iii) the fraction of [3H]DNA excised by exonuclease III was reduced in proportion to the actual length of the radiolabeled DNA; (iv) the effects of the two exonucleases were additive, consistent with each enzyme trimming only the 3' or 5' ends of nascent DNA chains without continued excision through to the opposite end. When the fraction of nascent [3H]DNA excised from replicating SV40 DNA by exonuclease III was compared with the fraction of [32P]DNA simultaneously excised from an SV40 DNA restriction fragment, the actual length of nascent [3H]DNA was calculated. From this number, the fraction of [3H]DNA excised from replicating SV40 chromosomes was converted into the number of nucleotides. Accordingly, the average distance from either 3' or 5' ends of long nascent DNA chains to the first nucleosome on either arm of replication forks was found to be 125 nucleotides. Furthermore, each exonuclease excised about 80% of the radiolabel in Okazaki fragments, suggesting that less than one-fifth of the Okazaki fragments were contained in nucleosomes. On the basis of these and other results, a model for eukaryotic replication forks is presented in which nucleosomes appear rapidly on both the forward and retrograde arms, about 125 and 300 nucleotides, respectively, from the actual site of DNA synthesis. In addition, it is proposed that Okazaki fragments are initiated on nonnucleosomal DNA and then assembled into nucleosomes, generally after ligation to the 5' ends of long nascent DNA chains is completed.     

Mechanism of DNA polymerase alpha inhibition by aphidicolin

Synthetic oligonucleotides of defined sequence were used to examine the mechanism of calf thymus DNA polymerase alpha inhibition by aphidicolin. Aphidicolin competes with each of the four dNTPs for binding to a pol alpha-DNA binary complex and thus should not be viewed as a dCTP analogue. Kinetic evidence shows that inhibition proceeds through the formation of a pol alpha.DNA.aphidicolin ternary complex, while DNase I protection experiments provide direct physical evidence. When deoxyguanosine is the next base to be replicated, Ki = 0.2 microM. In contrast, the Ki is 10-fold higher when the other dNMPs are at this position. Formation of a pol alpha.DNA.aphidicolin ternary complex did not inhibit the primase activity of the pol alpha.primase complex. Neither the rate of primer synthesis nor the size distribution of primers 2-10 nucleotides long was changed. Elongation of the primase-synthesized primers by pol alpha was inhibited both by ternary complex formation using exogenously added DNA and by aphidicolin alone.     

Replication protein A modulates its interface with the primed DNA template during RNA-DNA primer elongation in replicating SV40 chromosomes

The eukaryal single-stranded DNA binding protein replication protein A (RPA) binds short oligonucleotides with high affinity but exhibits low cooperativity in binding longer templates, opposite to prokaryal counterparts. This discrepancy could reflect the smaller size of the replicative template portion availed to RPA. According to current models, this portion accommodates an RNA–DNA primer (RDP) of <40 nt (nested discontinuity) or a several-fold longer Okazaki fragment (initiation zone). Previous in situ UV-crosslinking revealed that RPA also interacts with nascent DNA, especially growing RDPs. Here we compare nascent SV40 DNA chains UV-crosslinked to the middle and large RPA subunits and use the data to re-examine the two models. The middle subunit interacted with the nascent chains after a few DNA residues were added to the RNA primer while the large subunit became accessible after extension by several more. Upon RDP maturation, the middle subunit disengaged while the large subunit remained accessible during further limited extension. A corresponding shift in preference in favor of the large subunit has been reported for purified RPA and synthetic gapped duplexes upon reduction of the gap from 19 to 9 nt. Combined, these facts support the proposal that the mature RDP faces downstream a correspondingly small gap, possibly created by removal of the RNA primer moiety from an adjacent, previously synthesized RDP (nested discontinuity) but insufficient for continuous elongation of the RDP into an Okazaki fragment (initiation zone).     

Influence of poly(ADP-ribose) polymerase on the enzymatic synthesis of SV40 DNA.

The influence of poly(ADP-ribose) polymerase (PARP) on the replication of DNA containing the SV40 origin of replication has been examined. Extensive replication of SV40 DNA can be carried out in the presence of T antigen, topoisomerase I, the multimeric human single strand DNA-binding protein (HSSB), and DNA polymerase alpha-DNA primase (pol alpha-primase) complex (the monopolymerase system). In the monopolymerase system, both small products (Okazaki fragments), arising from lagging strand synthesis, and long products, arising from leading strand synthesis, are formed. The synthesis of long products requires the presence of relatively high levels of pol alpha-primase complex. In the presence of PARP, the synthesis of long products was blocked and only small Okazaki fragments accumulated, arising from the replication of the lagging strand template. The inhibition of leading strand synthesis by PARP can be effectively reversed by supplementing the monopolymerase system with the multimeric activator 1 protein (A1), the proliferating cell nuclear antigen (PCNA) and PCNA-dependent DNA polymerase delta (the dipolymerase system). The inhibition of leading strand synthesis in the monopolymerase system was caused by the binding of PARP to the ends of DNA chains, which blocked their further extension by pol alpha. The selective accumulation of Okazaki fragments was shown to be due to the coupled synthesis of primers by DNA primase and their immediate extension by pol alpha complexed to primase. PARP had little effect on this coupled reaction, but did inhibit the subsequent elongation of products, presumably after pol alpha dissociated from the 3'-end of the DNA fragments. PARP inhibited several other enzymatic reactions which required free ends of DNA chains. PARP inhibited exonuclease III, DNA ligase, the 5' to 3' exonuclease, and the elongation of primed DNA templates by pol alpha. In contrast, PARP only partly competed with the elongation of primed DNA templates by the pol delta elongation system which required SSB, A1, and PCNA. These results suggest that the binding of PARP at the ends of nascent DNA chains can be displaced by the binding of A1 and PCNA to primer ends. HSSB can be poly(ADP-ribosylated) in vivo as well as in vitro. However, the selective effect of PARP in blocking leading strand synthesis in the monopolymerase system was shown to depend primarily on its DNA binding property rather than on its ability to synthesize poly(ADP-ribose).     

Eukaryotic DNA replication. Enzymes and proteins acting at the fork

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. model systems for replication, such as the simian virus 40 DNA, lead the way. Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double-helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase delta and the lagging strand discontinuously in small pieces by DNA polymerase alpha. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3'----5'-exonuclease, mostly part of the DNA polymerase delta polypeptide, can perform proof-reading by excising incorrectly base-paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5'----3'-exonuclease, removing the RNA primers, DNA polymerase alpha or beta, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher-order structures like chromatin.     

Involvement of eucaryotic deoxyribonucleic acid polymerases alpha and gamma in the replication of cellular and viral deoxyribonucleic acid.

In an effort to identify the deoxyribonucleic acid (DNA) polymerase activities responsible for mammalian viral and cellular DNA replication, the effect of DNA synthesis inhibitors on isolated DNA polymerases was compared with their effects on viral and cellular DNA replication in vitro. DNA polymerase alpha, simian virus 40 (SV40) DNA replication in nuclear extracts, and CV-1 cell (the host for SV40) DNA replication in isolated nuclei all responded to DNA synthesis inhibitors in a quantitatively similar manner: they were relatively insensitive to 2',3'-dideoxythymidine 5'-triphosphate (d2TTP), but completely inhibited by aphidicolin, 1-beta-D-arabinofuranosylcytosine 5'-triphosphate (araCTP), and N-ethylmaleimide. In comparison, DNA polymerases beta and gamma were inhibited by d2TTP but insensitive to aphidicolin and 20--30 times less sensitive to araCTP than DNA polymerase alpha. Herpes simplex virus type 1 (HSV-1) DNA polymerase and DNA polymerase alpha were the only enzymes tested that were relatively insensitive to d2TTP; DNA polymerases beta and gamma, phage T4 and T7 DNA polymerases, and Escherichia coli DNA polymerase I were 100--250 times more sensitive. The results with d2TTP were independent of enzyme concentration, primer-template concentration, primer-template choice, and the labeled dNTP. A specific requirement for DNA polymerase alpha in the replication of SV40 DNA was demonstrated by the fact that DNA polymerase alpha was required, in addition to other cytosol proteins, to reconstitute SV40 DNA replication activity in N-ethylmaleimide-inactivated nuclear extracts containing replicating SV40 chromosomes. DNA polymerases beta and gamma did not substitute for DNA polymerase alpha. In contrast to SV40 and CV-1 DNA replication, adenovirus type 2 (Ad-2) DNA replication in isolated nuclei was inhibited by d2TTP to the same extent as gamma-polymerase. Ad-2 DNA replication was also inhibited by aphidicolin to the same extent as alpha-polymerase. Synthesis of CV-1 DNA, SV40 DNA, and HSV-1 DNA in intact CV-1 cells was inhibited by aphidicolin. Ad-2 DNA replication was also inhibited, but only at a 100-fold higher concentration. We found no effect of 2'-3'-dideoxythymidine (d2Thd) on cellular or viral DNA replication in spite of the fact that Ad-2 DNA replication in isolated nuclei was inhibited 50% by a ratio of d2TTP/dTTP of 0.02. This was due to the inability of CV-1 and Hela cells to phosphorylate d2Thd to d2TTP. These data are consistent with the hypothesis that DNA polymerase alpha is the only DNA polymerase involved in replicating SV40 DNA and CV-1 DNA and that Ad-2 DNA replication involves both DNA polymerases gamma and alpha.     

Sequence specificity for the initiation of RNA-primed simian virus 40 DNA synthesis in vivo

Analysis of the nucleotide sequences at the 5' ends of RNA-primed nascent DNA chains (Okazaki fragments) and of their locations in replicating simian virus 40 (SV40) DNA revealed the precise nature of Okazaki fragment initiation sites in vivo. The primary initiation site for mammalian DNA primase was 3'-purine-dT-5' in the DNA template and the secondary site was 3'-purine-dC-5', with the 5' end of the RNA primer complementary to either the dT or dC. The third position of the initiation site was variable with a preference for dT or dA. About 81% of the available 3'-purine-dT-5' sites and 20% of the 3'-purine-dC-5' sites were used. Purine-rich sites, such as PuPuPu and PyPuPu , were excluded. The 5'-terminal ribonucleotide composition of Okazaki fragments corroborated these conclusions. Furthermore, the length of individual RNA primers was not unique, but varied in size from six to ten bases with some appearing as short as three bases and some as long as 12 bases, depending on the initiation site used. This result was consistent with the average size (9 to 11 bases) of RNA primers isolated from specific regions of the genome. Excision of RNA primers did not appear to stop at the RNA-DNA junction, but removed a variable number of deoxyribonucleotides from the 5' end of the nascent DNA chain. Finally, only one-fourth of the replication forks contained an Okazaki fragment, and the distribution of their initiation sites between the two arms revealed that Okazaki fragments were initiated exclusively (99%) on retrograde DNA templates. The data obtained at two genomic sites about 350 and 1780 bases from ori were essentially the same as that reported for the ori region (Hay & DePamphilis , 1982), suggesting that the mechanism used to synthesize the first DNA chain at ori is the same as that used to synthesize Okazaki fragments throughout the genome.