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.     

An Okazaki piece of simian virus 40 may be synthesized by ligation of shorter precursor chains.

It is generally accepted that an aphidicolin-sensitive DNA polymerase elongates the eucaryotic RNA primer (iRNA) into a mature Okazaki piece reaching ca. 200 nucleotides. Yet, as shown here, nascent DNA chains below 40 nucleotides accumulated in simian virus 40 (SV40) DNA replicating in isolated nuclei in the presence of aphidicolin. These products resembled precursors of longer Okazaki pieces synthesized in the absence of aphidicolin (termed here DNA primers) in size distribution, lagging-replication-fork polarity, and content of iRNA. Within the isolated SV40 replicative intermediate, DNA primers could be extended in a reaction catalyzed by the Escherichia coli DNA polymerase I large fragment. This increased their length by an average of 21 deoxyribonucleotide residues, indicating that single-stranded gaps of corresponding length existed 3' to the DNA primers. Incubation with T4 DNA ligase converted most of the extended DNA primers into products resembling long Okazaki pieces. These data led us to propose that the synthesis of an SV40 Okazaki piece could be itself discontinuous and could comprise the following steps: (i) iRNA synthesis by DNA primase, (ii) iRNA extension into a DNA primer by an aphidicolin-resistant activity associated with DNA primase-DNA polymerase alpha, (iii) removal of iRNA moieties between adjacent DNA primers, (iv) "gap filling" between DNA primers by the aphidicolin-sensitive DNA polymerase alpha, and (v) ligation of DNA primer units onto a growing Okazaki piece. Eventually, a mature Okazaki piece is ligated onto a longer nascent DNA chain.     

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.     

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.     

Evidence that errors made by DNA polymerase alpha are corrected by DNA polymerase delta

Eukaryotic replication begins at origins and on the lagging strand with RNA-primed DNA synthesis of a few nucleotides by polymerase alpha, which lacks proofreading activity. A polymerase switch then allows chain elongation by proofreading-proficient pol delta and pol epsilon. Pol delta and pol epsilon are essential, but their roles in replication are not yet completely defined . Here, we investigate their roles by using yeast pol alpha with a Leu868Met substitution . L868M pol alpha copies DNA in vitro with normal activity and processivity but with reduced fidelity. In vivo, the pol1-L868M allele confers a mutator phenotype. This mutator phenotype is strongly increased upon inactivation of the 3' exonuclease of pol delta but not that of pol epsilon. Several nonexclusive explanations are considered, including the hypothesis that the 3' exonuclease of pol delta proofreads errors generated by pol alpha during initiation of Okazaki fragments. Given that eukaryotes encode specialized, proofreading-deficient polymerases with even lower fidelity than pol alpha, such intermolecular proofreading could be relevant to several DNA transactions that control genome stability.     

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.     

Double-strand break repair in yeast requires both leading and lagging strand DNA polymerases

Mitotic double-strand break (DSB)-induced gene conversion at MAT in Saccharomyces cerevisiae was analyzed molecularly in mutant strains thermosensitive for essential replication factors. The processivity cofactors PCNA and RFC are essential even to synthesize as little as 30 nucleotides following strand invasion. Both PCNA-associated DNA polymerases delta and epsilon are important for gene conversion, though a temperature-sensitive Pol epsilon mutant is more severe than one in Pol delta. Surprisingly, mutants of lagging strand replication, DNA polymerase alpha (pol1-17), DNA primase (pri2-1), and Rad27p (rad27 delta) also greatly inhibit completion of DSB repair, even in G1-arrested cells. We propose a novel model for DSB-induced gene conversion in which a strand invasion creates a modified replication fork, involving leading and lagging strand synthesis from the donor template. Replication is terminated by capture of the second end of the DSB.     

Lagging strand DNA synthesis by calf thymus DNA polymerases alpha, beta, delta and epsilon in the presence of auxiliary proteins.

By using a defined gapped DNA substrate that mimics a lagging strand of 230 nucleotides and that contains a defined pause site, we have analyzed calf thymus DNA polymerases (pol) alpha, beta, delta, and epsilon in the presence of the three auxiliary proteins proliferating cell nuclear antigen (PCNA), replication factor C (RF-C) and replication protein A (RP-A) for their ability to complete an Okazaki fragment. Pol alpha alone could fill the gap to near completion, but was strongly stopped by the pause site. Addition of low amounts of RP-A resulted in an increased synthesis by pol alpha past the pause site. In contrast, high amounts of RP-A strongly inhibited gap filling by pol alpha. Further inhibition was evident when the two other auxiliary proteins, PCNA and RF-C, were added in addition to RP-A. Pol beta could completely fill the gap without specific pausing and also was strongly inhibited by RP-A. PCNA and RF-C had no detectable effect on pol beta. Pol delta, relied as expected, on all three auxiliary proteins for complete gap filling synthesis and could, upon longer incubation, perform a limited amount of strand displacement synthesis. Pol epsilon core enzyme was able to fill the gap completely, but like pol alpha, essentially stopped at the pause site. This pausing could only be overcome upon addition of PCNA, RF-C and E. coli single-stranded DNA binding protein. Thus pol epsilon holoenzyme preferentially synthesized to the end of the gap without pausing. Ligation of the DNA products indicated that pol beta core enzyme, pol delta and pol epsilon holoenzymes (but not pol alpha and pol epsilon core enzyme) synthesized products that were easily ligatable. Our results indicate that pol epsilon holoenzyme fills a defined lagging strand gapped template to exact completion and is able to pass a pause site. The data favour the hypothesis of Burgers (Burgers, P.M.J. (1991) J. Biol. Chem. 266, 22698-22706) that pol epsilon might be a candidate for the second replication enzyme at the lagging strand of the replication fork.     

DNA polymerase delta is highly processive with proliferating cell nuclear antigen and undergoes collision release upon completing DNA

In most cells, 100-1000 Okazaki fragments are produced for each replicative DNA polymerase present in the cell. For fast-growing cells, this necessitates rapid recycling of DNA polymerase on the lagging strand. Bacteria produce long Okazaki fragments (1-2 kb) and utilize a highly processive DNA polymerase III (pol III), which is held to DNA by a circular sliding clamp. In contrast, Okazaki fragments in eukaryotes are quite short, 100-250 bp, and thus the eukaryotic lagging strand polymerase does not require a high degree of processivity. The lagging strand polymerase in eukaryotes, polymerase delta (pol delta), functions with the proliferating cell nuclear antigen (PCNA) sliding clamp. In this report, Saccharomyces cerevisiae pol delta is examined on model substrates to gain insight into the mechanism of lagging strand replication in eukaryotes. Surprisingly, we find pol delta is highly processive with PCNA, over at least 5 kb, on Replication Protein A (RPA)-coated primed single strand DNA. The high processivity of pol delta observed in this report contrasts with its role in synthesis of short lagging strand fragments, which require it to rapidly dissociate from DNA at the end of each Okazaki fragment. We find that this dilemma is solved by a "collision release" process in which pol delta ejects from PCNA upon extending a DNA template to completion and running into the downstream duplex. The released pol delta transfers to a new primed site, provided the new site contains a PCNA clamp. Additional results indicate that the collision release mechanism is intrinsic to the pol3/pol31 subunits of the pol delta heterotrimer.     

Coordinated leading- and lagging-strand synthesis at the Escherichia coli DNA replication fork. V. Primase action regulates the cycle of Okazaki fragment synthesis.

Replication forks formed during rolling-circle DNA synthesis supported by a tailed form II DNA substrate in the presence of the primosome, the single-stranded DNA binding protein, and the DNA polymerase III holoenzyme (Pol III HE) that had been reconstituted from the purified subunits, beta, tau, and the gamma.delta complex, at limiting (with respect to nucleotide incorporation) concentrations of the Pol III core (alpha, epsilon, and theta) produced aberrantly small Okazaki fragments, while the synthesis of the leading strand was unperturbed. These small Okazaki fragments were not arrayed in tandem along the lagging-strand DNA template, but were separated by large gaps. Similarly structured synthetic products were not manufactured by replication forks reconstituted with higher, saturating concentrations of the Pol III core. Replication forks producing these small fragments could respond, by modulating the size of the Okazaki fragments produced, to variations in the concentration of NTPs or the primase, conditions that affect the frequency of priming on the lagging strand, but not to variation in the concentration of dNTPs, conditions that affect the frequency of utilization of the primers. Significantly longer Okazaki fragments (greater than 7 kilobases) could be produced in the presence of a limiting amount of Pol III core at low concentrations of the primase. These observations indicated that the production of small Okazaki fragments was not a result of a debilitated lagging-strand Pol III core, but rather a function of the time available for nascent strand synthesis during the cycle of events that are required for the manufacture of an Okazaki fragment and that it was the association of primase with the replication fork that keyed this cycle.     

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).     

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.     

Synthesis of DNA by DNA polymerase epsilon in vitro.

The isolation of DNA polymerase (Pol) epsilon from extracts of HeLa cells is described. The final fractions contained two major subunits of 210 and 50 kDa which cosedimented with Pol epsilon activity, similar to those described previously (Syvaoja, J., and Linn, S. (1989) J. Biol. Chem. 264, 2489-2497). The properties of the human Pol epsilon and the yeast Pol epsilon were compared. Both enzymes elongated singly primed single-stranded circular DNA templates. Yeast Pol epsilon required the presence of a DNA binding protein (SSB) whereas human Pol epsilon required the addition of SSB, Activator 1 and proliferating cell nuclear antigen (PCNA) for maximal activity. Both enzymes were totally unable to elongate primed DNA templates in the presence of salt; however, activity could be restored by the addition of Activator 1 and PCNA. Like Pol delta, Pol epsilon formed complexes with SSB-coated primed DNA templates in the presence of Activator 1 and PCNA which could be isolated by filtration through Bio-Gel A-5m columns. Unlike Pol delta, Pol epsilon bound to SSB-coated primed DNA in the absence of the auxiliary factors. In the presence of salt, Pol epsilon complexes were less stable than they were in the absence of salt. In the in vitro simian virus 40 (SV40) T antigen-dependent synthesis of DNA containing the SV40 origin of replication, yeast Pol epsilon but not human Pol epsilon could substitute for yeast or human Pol delta in the generation of long DNA products. However, human Pol epsilon did increase slightly the length of DNA chains formed by the DNA polymerase alpha-primase complex in SV40 DNA synthesis. The bearing of this observation on the requirement for a PCNA-dependent DNA polymerase in the synthesis and maturation of Okazaki fragments is discussed. However, no unique role for human Pol epsilon in the in vitro SV40 DNA replication system was detected.     

Assembly of DNA polymerase delta and epsilon holoenzymes depends on the geometry of the DNA template.

To study in details the assembly of DNA polymerases delta and epsilon holoenzymes a circular double-stranded DNA template containing a gap of 45 nucleotides was constructed. Both replication factor C and proliferating cell nuclear antigen were absolutely required and sufficient for assembly of DNA polymerase delta holoenzyme complex on DNA. On such a circular DNA substrate replication protein A (or E. coli single-strand DNA binding protein) was neither required for assembly of DNA polymerase delta holoenzyme complex nor for the gap-filling reaction. A circular structure of the DNA substrate was found to be absolutely critical for the ability of auxiliary proteins to interact with DNA polymerases. The linearization of the circular DNA template resulted in three dramatic effects: (i) DNA synthesis by DNA polymerase delta holoenzyme was abolished, (ii) the inhibition effect of replication factor C and proliferating cell nuclear antigen on DNA polymerase alpha was relieved and (iii) DNA polymerase epsilon could not form any longer a holoenzyme with replication factor C and proliferating cell nuclear antigen. The comparison of the effect of replication factor C and proliferating cell nuclear antigen on DNA polymerases alpha, delta and epsilon indicated that the auxiliary proteins appear to form a mobile clamp, which can easily slide along double-stranded DNA.     

Lagging strand synthesis in coordinated DNA synthesis by bacteriophage t7 replication proteins

The proteins of bacteriophage T7 DNA replication mediate coordinated leading and lagging strand synthesis on a minicircle template. A distinguishing feature of the coordinated synthesis is the presence of a replication loop containing double and single-stranded DNA with a combined average length of 2600 nucleotides. Lagging strands consist of multiple Okazaki fragments, with an average length of 3000 nucleotides, suggesting that the replication loop dictates the frequency of initiation of Okazaki fragments. The size of Okazaki fragments is not affected by varying the components (T7 DNA polymerase, gene 4 helicase-primase, gene 2.5 single-stranded DNA binding protein, and rNTPs) of the reaction over a relatively wide range. Changes in the size of Okazaki fragments occurs only when leading and lagging strand synthesis is no longer coordinated. The synthesis of each Okazaki fragment is initiated by the synthesis of an RNA primer by the gene 4 primase at specific recognition sites. In the absence of a primase recognition site on the minicircle template no lagging strand synthesis occurs. The size of the Okazaki fragments is not affected by the number of recognition sites on the template.     

Total reconstitution of DNA polymerase III holoenzyme reveals dual accessory protein clamps

DNA polymerase III holoenzyme (holoenzyme) is the 10-subunit replicase of the Escherichia coli chromosome. In this report, pure preparations of delta, delta', and a gamma chi psi complex are resolved from the five protein gamma complex subassembly. Using these subunits and other holoenzyme subunits isolated from overproducing plasmid strains of E. coli, the rapid and highly processive holoenzyme has been reconstituted from only five pure single subunits: alpha, epsilon, gamma, delta, and beta. The preceding report showed that of the three subunits in the core polymerase, only a complex of alpha (DNA polymerase) and epsilon (3'-5' exonuclease) are required to assemble a processive holoenzyme on a template containing a preinitiation complex (Studwell, P.S., and O'Donnell, M. (1990) J. Biol. Chem. 265, 1171-1178). This report shows that of the five proteins in the gamma complex only a heterodimer of gamma and delta is required with the beta subunit to form the ATP-activated preinitiation complex with a primed template. Surprisingly, the delta' subunit does not form an active complex with gamma but forms a fully active heterodimer complex with the tau subunit (as does delta). Hence, the tau delta' and gamma delta heterodimers are fully active in the preinitiation complex reaction with beta and primed DNA. Holoenzymes reconstituted using the alpha epsilon complex, beta subunit, and either gamma delta or tau delta' are fully processive in DNA synthesis, and upon completing the template they rapidly cycle to a new primed template endowed with a preinitiation complex clamp. Since the holoenzyme molecule contains all of these accessory subunits (gamma, delta, tau, delta', and beta) in all likelihood it has the capacity to form two preinitiation complex clamps simultaneously at two primer termini. Two primer binding components within one holoenzyme may mediate its rapid cycling to multiple primers on the lagging strand and also provides functional evidence for the hypothesis of holoenzyme as a dimeric polymerase capable of simultaneous replication of both leading and lagging strands of a replication fork.     

Fidelity of mammalian DNA replication and replicative DNA polymerases.

Current models suggest that two or more DNA polymerases may be required for high-fidelity semiconservative DNA replication in eukaryotic cells. In the present study, we directly compare the fidelity of SV40 origin-dependent DNA replication in human cell extracts to the fidelity of mammalian DNA polymerases alpha, delta, and epsilon using lacZ alpha of M13mp2 as a reporter gene. Their fidelity, in decreasing order, is replication greater than or equal to pol epsilon greater than pol delta greater than pol alpha. DNA sequence analysis of mutants derived from extract reactions suggests that replication is accurate when considering single-base substitutions, single-base frameshifts, and larger deletions. The exonuclease-containing calf thymus DNA polymerase epsilon is also highly accurate. When high concentrations of deoxynucleoside triphosphates and deoxyguanosine monophosphate are included in the pol epsilon reaction, both base substitution and frameshift error rates increase. This response suggests that exonucleolytic proofreading contributes to the high base substitution and frameshift fidelity. Exonuclease-containing calf thymus DNA polymerase delta, which requires proliferating cell nuclear antigen for efficient synthesis, is significantly less accurate than pol epsilon. In contrast to pol epsilon, pol delta generates errors during synthesis at a relatively modest concentration of deoxynucleoside triphosphates (100 microM), and the error rate did not increase upon addition of adenosine monophosphate. Thus, we are as yet unable to demonstrate that exonucleolytic proofreading contributes to accuracy during synthesis by DNA polymerase delta. The four-subunit DNA polymerase alpha-primase complex from both HeLa cells and calf thymus is the least accurate replicative polymerase. Fidelity is similar whether the enzyme is assayed immediately after purification or after being stored frozen.(ABSTRACT TRUNCATED AT 250 WORDS)     

Replication factors required for SV40 DNA replication in vitro. II. Switching of DNA polymerase alpha and delta during initiation of leading and lagging strand synthesis.

Replication factors A and C (RF-A and RF-C) and the proliferating cell nuclear antigen (PCNA) differentially augment the activities of DNA polymerases alpha and delta. The mechanism of stimulation by these replication factors was investigated using a limiting concentration of primed, single-stranded template DNA. RF-A stimulated polymerase alpha activity in a concentration-dependent manner, but also suppressed nonspecific initiation of DNA synthesis by both polymerases alpha and delta. The primer recognition complex, RF-C.PCNA.ATP, stimulated pol delta activity in cooperation with RF-A, but also functioned to prevent abnormal initiation of DNA synthesis by polymerase alpha. Reconstitution of DNA replication with purified factors and a plasmid containing the SV40 origin sequences directly demonstrated DNA polymerase alpha dependent synthesis of lagging strands and DNA polymerase delta/PCNA/RF-C dependent synthesis of leading strands. RF-A and the primer recognition complex both affected the relative levels of leading and lagging strands. These results, in addition to results in an accompanying paper (Tsurimoto, T., and Stillman, B. (1991) J. Biol. Chem. 266, 1950-1960), suggest that an exchange of DNA polymerase complexes occurs during initiation of bidirectional DNA replication at the SV40 origin.     

Induction of DNA polymerase activities in the regenerating rat liver.

The levels of DNA polymerase alpha, DNA polymerase delta, and its accessory protein, proliferating cell nuclear antigen (PCNA) were examined in the regenerating rat liver. The levels of DNA polymerase alpha and delta activities in regenerating liver extracts were determined by the use of the DNA polymerase alpha specific inhibitor, BuAdATP [2-(p-n-butylanilino)-9-(2-deoxy-beta-D-ribofuranosyl) adenine 5'-triphosphate], and monoclonal antibodies. These reagents showed that the total DNA polymerase activities increased ca. 4-fold during regeneration and that the fraction of DNA polymerase delta activity at the peak was 40% of the total DNA polymerase activity. Immunoblots and inhibition studies using specific antibodies showed that DNA polymerase delta and epsilon and PCNA were concomitantly induced after partial hepatectomy. The levels of both DNA polymerase delta and epsilon and PCNA reached their maxima at 24-36 h post hepatectomy, i.e., at the same time that in vivo DNA synthesis reached its peak. Partial purification and characterization of DNA polymerases delta and epsilon from the regenerating rat liver were also performed. These observations suggest that the variation of DNA polymerase delta and epsilon and PCNA during liver regeneration is closely related to DNA synthesis and is consistent with their involvement in DNA replication.     

DNA polymerase epsilon: the latest member in the family of mammalian DNA polymerases

DNA polymerase epsilon is a mammalian polymerase that has a tightly associated 3'----5' exonuclease activity. Because of this readily detectable exonuclease activity, the enzyme has been regarded as a form of DNA polymerase delta, an enzyme which, together with DNA polymerase alpha, is in all probability required for the replication of chromosomal DNA. Recently, it was discovered that DNA polymerase epsilon is both catalytically and structurally distinct from DNA polymerase delta. The most striking difference between the two DNA polymerases is that processive DNA synthesis by DNA polymerase delta is dependent on proliferating cell nuclear antigen (PCNA), a replication factor, while DNA polymerase epsilon is inherently processive. DNA polymerase epsilon is required at least for the repair synthesis of UV-damaged DNA. DNA polymerases are highly conserved in eukaryotic cells. Mammalian DNA polymerases alpha, delta and epsilon are counterparts of yeast DNA polymerases I, III and II, respectively. Like DNA polymerases I and III, DNA polymerase II is also essential for the viability of cells, which suggests that DNA polymerase II (and epsilon) may play a role in DNA replication.