Pol Alpha-primase Research Articles

Mechanism of cell cycle arrest by (8E,13Z,20Z)-strobilinin/(7E,13Z,20Z)-felixinin from a marine sponge Psammocinia sp.

Furanosesterterpenes, isolated from a marine sponge Psammocinia sp. have been reported to display significant cytotoxicity to some cancer cell lines. In this study, EZZ, an inseparable 1:1 mixture of (8E,3Z,20Z)-strobilinin and (7E,3Z,20Z)-felixinin, showed significant antiproliferative effect on human cervix carcinoma cell line (HeLa). Cell cycle analysis revealed that EZZ could arrest HeLa cells in S phase with a concomitant decrease in the cell population of G1 phase. By using simian virus (SV40) DNA in vitro replication system, we found that EZZ could inhibit DNA replication, which suggests that EZZ-induced S phase arrest might be the direct result of blocked DNA synthesis. Furthermore, low concentration of EZZ was found to be capable of significantly inhibiting the DNA cleavage by topoisomerase I (topo I) and reducing the polymerase alpha-primase (pol alpha-primase) activity, while the ssDNA binding activity of replication protein A (RPA) was less affected. Taken together, these results suggest that EZZ-induced cell cycle arrest in S phase correlate with the inhibition of DNA replication, and topo I and pol alpha-primase might be the two main target molecules.

Role of DNA replication proteins in double-strand break-induced recombination in Saccharomyces cerevisiae.

Mitotic double-strand break (DSB)-induced gene conversion involves new DNA synthesis. We have analyzed the requirement of several essential replication components, the Mcm proteins, Cdc45p, and DNA ligase I, in the DNA synthesis of Saccharomyces cerevisiae MAT switching. In an mcm7-td (temperature-inducible degron) mutant, MAT switching occurred normally when Mcm7p was degraded below the level of detection, suggesting the lack of the Mcm2-7 proteins during gene conversion. A cdc45-td mutant was also able to complete recombination. Surprisingly, even after eliminating both of the identified DNA ligases in yeast, a cdc9-1 dnl4 Delta strain was able to complete DSB repair. Previous studies of asynchronous cultures carrying temperature-sensitive alleles of PCNA, DNA polymerase alpha (Pol alpha), or primase showed that these mutations inhibited MAT switching (A. M. Holmes and J. E. Haber, Cell 96:415-424, 1999). We have reevaluated the roles of these proteins in G(2)-arrested cells. Whereas PCNA was still essential for MAT switching, neither Pol alpha nor primase was required. These results suggest that arresting cells in S phase using ts alleles of Pol alpha-primase, prior to inducing the DSB, sequesters some other component that is required for repair. We conclude that DNA synthesis during gene conversion is different from S-phase replication, involving only leading-strand polymerization.

Primer Utilization by DNA Polymerase α-Primase Is Influenced by Its Interaction with Mcm10p

Models of DNA replication in yeast and Xenopus suggest that Mcm10p is required to generate the pre-initiation complex as well as progression of the replication fork during the elongation of DNA chains. In this report, we show that the Schizosaccharomyces pombe Mcm10p/Cdc23p binds to the S. pombe DNA polymerase (pol) alpha-primase complex in vitro by interacting specifically with the catalytic p180 subunit and stimulates DNA synthesis catalyzed by the pol alpha-primase complex with various primed DNA templates. We investigated the mechanism by which Mcm10p activates the polymerase activity of the pol alpha-primase complex by generating truncated derivatives of the full-length 593-amino acid Mcm10p. Their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA and to pol alpha were compared. Concomitant with increased deletion of the N-terminal region (from amino acids 95 to 415), Mcm10p derivatives lost their ability to stimulate pol alpha polymerase activity and bind to single-stranded DNA. Truncated derivatives of Mcm10p containing amino acids 1-416 retained the pol alpha binding activity, whereas the C terminus, amino acids 496-593, did not. These results demonstrate that both the single-stranded DNA binding and the pol alpha binding properties of Mcm10p play important roles in the activation. In accord with these findings, Mcm10p facilitated the binding of pol alpha-primase complex to primed DNA and formed a stable complex with pol alpha-primase on primed templates. A mutant that failed to activate or bind to DNA and pol alpha, was not observed in this complex. We suggest that the interaction of Mcm10p with the pol alpha-primase complex, its binding to single-stranded DNA, and its activation of the polymerase complex together contribute to its role in the elongation phase of DNA replication.

Dissociation of DNA polymerase alpha-primase complex during meiosis in Coprinus cinereus.

Previously, the activity of DNA polymerase alpha was found in the meiotic prophase I including non-S phase stages, in the basidiomycetes, Coprinus cinereus. To study DNA polymerase alpha during meiosis, we cloned cDNAs for the C. cinereus DNA polymerase alpha catalytic subunit (p140) and C. cinereus primase small subunit (p48). Northern analysis indicated that both p140 and p48 are expressed not only at S phase but also during the leptotene/zygotene stages of meiotic prophase I. In situ immuno-staining of cells at meiotic prophase I revealed a sub population of p48 that does not colocalize with p140 in nuclei. We also purified the pol alpha-primase complex from meiotic cells by column chromatography and characterized its biochemical properties. We found a subpopulation of primase that was separated from the pol alpha-primase complex by phosphocellulose column chromatography. Glycerol gradient density sedimentation results indicated that the amount of intact pol alpha-primase complex in crude extract is reduced, and that a smaller complex appears upon meiotic development. These results suggest that the form of the DNA polymerase alpha-primase complex is altered during meiotic development.

Replication Protein A as a “Fidelity Clamp” for DNA Polymerase α

The current view of DNA replication in eukaryotes predicts that DNA polymerase alpha (pol alpha)-primase synthesizes the first 10-ribonucleotide-long RNA primer on the leading strand and at the beginning of each Okazaki fragment on the lagging strand. Subsequently, pol alpha elongates such an RNA primer by incorporating about 20 deoxynucleotides. pol alpha displays a low processivity and, because of the lack of an intrinsic or associated 3'--> 5' exonuclease activity, it is more error-prone than other replicative pols. Synthesis of the RNA/DNA primer catalyzed by pol alpha-primase is a critical step in the initiation of DNA synthesis, but little is known about the role of the DNA replication accessory proteins in its regulation. In this paper we provide evidences that the single-stranded DNA-binding protein, replication protein A (RP-A), acts as an auxiliary factor for pol alpha playing a dual role: (i) it stabilizes the pol alpha/primer complex, thus acting as a pol clamp; and (ii) it significantly reduces the misincorporation efficiency by pol alpha. Based on these results, we propose a hypothetical model in which RP-A is involved in the regulation of the early events of DNA synthesis by acting as a "fidelity clamp" for pol alpha.

Species specificity of human RPA in simian virus 40 DNA replication lies in T-antigen-dependent RNA primer synthesis.

Replication protein A (RPA) is a three-subunit protein complex with multiple functions in DNA replication. Previous study indicated that human RPA (h-RPA) could not be replaced by Schizosaccharomyces pombe RPA (sp-RPA) in simian virus 40 (SV40) replication, suggesting that h-RPA may have a specific function in SV40 DNA replication. To understand the specificity of h-RPA in replication, we prepared heterologous RPAs containing the mixture of human and S.pombe subunits and compared these preparations for various enzymatic activities. Heterologous RPAs containing two human subunits supported SV40 DNA replication, whereas those containing only one human subunit poorly supported DNA replication, suggesting that RPA complex requires at least two human subunits to support its function in SV40 DNA replication. All heterologous RPAs effectively supported single-stranded (ss)DNA binding activity and an elongation of a primed DNA template catalyzed by DNA polymerase (pol) alpha and delta. A strong correlation between SV40 DNA replication activity and large tumor antigen (T-ag)-dependent RNA primer synthesis by pol alpha-primase complex was observed among the heterologous RPAs. Furthermore, T-ag showed a strong interaction with 70- and 34-kDa subunits from human, but poorly interacted with their S.pombe counterparts, indicating that the specificity of h-RPA is due to its role in RNA primer synthesis. In the SV40 replication reaction, the addition of increasing amounts of sp-RPA in the presence of fixed amount of h-RPA significantly reduced overall DNA synthesis, but increased the size of lagging strand, supporting a specific role for h-RPA in RNA primer synthesis. Together, these results suggest that the specificity of h-RPA in SV40 replication lies in T-ag-dependent RNA primer synthesis.

Activity of DNA polymerase alpha in aging human fibroblasts.

During the past decade intense investigation has focused on cellular aging with the expectation of discovering factors that regulate the replication complex and contribute to the onset and progression of cellular aging. The most striking feature of cellular aging is the failure of sensing diploid cells to enter or complete S phase of the cell cycle. The G1/S phase transition is an initial critical step in the regulation of proliferation in eukaryotic cells, and significant advances have been made toward understanding the basic mechanisms of aging by identifying components of the macromolecular assemblies participating in the G1/S transition. These studies have identified multiple DNA polymerases and their accessory factors, and have provided important strategies for investigating the molecular events that contribute to aging processes. DNA replication, repair and recombination in eukaryotic cells require the action of a variety of DNA polymerases, at least six of which are known, alpha, beta, gamma, delta, epsilon, and zeta. Among them the highly conserved DNA polymerase alpha-primase (pol alpha-primase) is the only enzyme capable of initiating DNA replication at chromosomal origin sites and at sites of initiation of discontinuous synthesis of Okazaki fragments on the lagging side of the replication fork. Numerous protein factors that play strategic roles in DNA replication have been identified and the understanding of their regulation has been an important step for identifying the elements that are involved in, and possibly necessary for, governing cellular senescence and aging. In this review we summarize the current information regarding DNA pol alpha modulation during aging. We focus in particular on the coordinated actions of DNA pol alpha in the presence of other cellular proteins involved in the replication complex in the hope that understanding pol alpha interactions with components of the replication complex may provide insight into the mechanisms by which aging and age-related diseases occur.

Abasic template lesions are strong chain terminators for DNA primase but not for DNA polymerase alpha during the synthesis of new DNA strands.

The effects of abasic lesions on both primase activity and DNA polymerase alpha- (pol alpha) catalyzed elongation of primase-synthesized primers were examined. Abasic lesions were strong chain terminators during primer synthesis by primase. However, extension of primase-synthesized primers by pol alpha resulted in 60-93% bypass of abasic lesions. Sequencing of bypass products generated during this primase-coupled pol alpha activity showed that dAMP was preferentially incorporated opposite the abasic lesion, indicating that pol alpha was responsible for bypass. In contrast, previous analyses of pol alpha-catalyzed elongation of exogenously supplied DNA primer-templates showed that abasic lesions strongly terminated DNA synthesis. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer-templates with respect to interaction with abasic lesions. Furthermore, this high level of abasic lesion bypass during primase-coupled pol alpha activity provides an additional mechanism for how translesional synthesis may occur in vivo, an event hypothesized to be mutagenic.

Interactions of DNA with human DNA primase monitored with photoactivatable cross-linking agents: implications for the role of the p58 subunit.

Regulation of the p49-p58 primase complex during primer synthesis and the interaction of the primase subunits with DNA were examined. After primase synthesizes a primer that DNA polymerase alpha (pol alpha) can readily elongate, further primase activity is negatively regulated. This occurs within both the context of the four-subunit pol alpha-primase complex and in the p49-p58 primase complex, indicating that the newly generated primer-template species need not interact with pol alpha to regulate further primase activity. Photo-cross-linking of single-stranded DNA-primase complexes revealed that whereas the isolated p49 and p58 subunits both reacted with DNA upon photolysis, only the p58 subunit reacted with the DNA when photolysis was performed using the p49-p58 primase complex. After primer synthesis by the complex, p58 was again the only subunit that reacted with the DNA. These results suggest a model for regulation of primer synthesis in which the newly synthesized primer-template species binds to p58 and regulates further primer synthesis. Additionally, the ability of p58 to interact with primer-template species suggests that p58 mediates the transfer of primers from the primase active site to pol alpha.

Replication protein-A mediates the association of calf thymus DNA polymerase alpha-DNA primase complex with guanine-rich DNA sequence.

We have shown that calf thymus DNA polymerase alpha-DNA primase complex (pol alpha-primase) preferentially binds to pyrimidine-rich sequences and initiates RNA primer synthesis [Suzuki, M. et al. (1993) Biochemistry 32, 12782-12792]. Here we tested the association of pol alpha-primase with a guanine-rich DNA fragment (SVG, 30-mer) containing in vivo initiation sites of simian virus 40 DNA replication. While pyrimidine-rich fragment (CTPPS 1, 30-mer), that is a preferred sequence for calf thymus DNA primase, was well co-precipitated with pol alpha-primase using anti-pol alpha antibody, SVG was hardly precipitated under the same conditions. Competition studies in either gel-retardation assay or during de novo DNA synthesis by pol alpha-primase demonstrated that the interaction of pol alpha-primase with SVG was much weaker than that with CTPPS-1. On the other hand, replication protein-A (RP-A) could bind SVG, although less efficiently than CTPPS 1. After preincubation with RP-A, SVG could bind pol alpha-primase that was immobilized on Sepharose beads. The simian virus 40 large T antigen also enhanced association of SVG to pol alpha-primase, while Escherichia coli single-stranded DNA-binding protein did not. However, pol alpha-primase, bound to SVG in the presence of RP-A, failed to synthesize RNA primers. When SVG was extended 10 nucleotides at its 5'-end, pol alpha-primase synthesized trace amounts of RNA primers, and this activity was stimulated more than 10-fold by adding RP-A. These results suggest a new role for RP-A, i.e., as a molecular tether that allows pol alpha-primase to bind guanine-rich regions of DNA in order to initiate RNA primer synthesis.

The Effects of Cytosine Arabinoside on RNA-primed DNA Synthesis by DNA Polymerase α-Primase

Oligonucleotides containing a specific initiation site for polymerase alpha-primase (pol alpha-primase) were used to measure the effects of cytosine arabinoside triphosphate and cytosine arabinoside monophosphate (araCMP) in DNA on RNA-primed DNA synthesis. Primase inserts araCMP at the 3' terminus of a full-length RNA primer with a 400-fold preference over CMP. The araCMP is elongated efficiently by pol alpha in the primase-coupled reaction. Extension from RNA 3'-araCMP is 50-fold less efficient than from CMP, and extension from DNA 3'-araCMP is 1600-fold less efficient than from dCMP. Using araCMP-containing templates, primer synthesis is reduced 2-3-fold, and RNA-primed DNA synthesis is reduced 2-8-fold. The efficiency of polymerization past a template araCMP by pol alpha is reduced 180-fold during insertion of dGMP opposite araCMP and 35-fold during extension from the araCMP:dGMP 3' terminus. These results show that the pol alpha-primase efficiently incorporates araCMP as the border nucleotide between RNA and DNA and suggest that the inhibitory effects of araC most likely result from slowed elongation of pol alpha and less so from inhibition of primer synthesis by primase.

Arabinofuranosyl nucleotides are not chain-terminators during initiation of new strands of DNA by DNA polymerase alpha-primase.

Polymerization of NTPs and arabinofuranosyladenosine triphosphate (araATP) during DNA polymerase alpha catalyzed elongation of primase-synthesized primers was examined. After primase synthesizes a primer, pol alpha normally polymerizes multiple dNTPs onto this primer. In the absence of a required dNTP, however, primers were still elongated by up to 35 nucleotides via polymerization of the corresponding NTP in place of the missing dNTP. During the elongation of exogenously added primer/templates, however, NTPs were not readily polymerized. AraATP was readily incorporated into products during elongation of primase-synthesized primers. Importantly, polymerization of araATP did not result in chain termination; rather, the next correct nucleotide was added such that araATP was simply an alternate substrate. In contrast, polymerization of araATP during elongation of exogenously added primer/templates resulted in strong chain termination. Thus, elongation of primase-synthesized primers by pol alpha-primase is fundamentally different than elongation of exogenously added primer/templates with respect to interactions with dNTP analogs. Furthermore, these data provide a rationale for how araNMPs are efficiently incorporated into internucleotide linkages of DNA in whole cells and suggest that the initiation of new strands of DNA by pol alpha-primase may be a unique target for inhibiting replication.

Initiation of RNA-primed DNA synthesis in vitro by DNA polymerase alpha-primase.

The initiation of new DNA strands at origins of replication in animal cells requires de novo synthesis of RNA primers by primase and subsequent elongation from RNA primers by DNA polymerase alpha. To study the specificity of primer site selection by the DNA polymerase alpha-primase complex (pol alpha-primase), a natural DNA template containing a site for replication initiation was constructed. Two single-stranded DNA (ssDNA) molecules were hybridized to each other generating a duplex DNA molecule with an open helix replication 'bubble' to serve as an initiation zone. Pol alpha-primase recognizes the open helix region and initiates RNA-primed DNA synthesis at four specific sites that are rich in pyrimidine nucleotides. The priming site positioned nearest the ssDNA-dsDNA junction in the replication 'bubble' template is the preferred site for initiation. Using a 40 base oligonucleotide template containing the sequence of the preferred priming site, primase synthesizes RNA primers of 9 and 10 nt in length with the sequence 5'-(G)GAAGAAAGC-3'. These studies demonstrate that pol alpha-primase selects specific nucleotide sequences for RNA primer formation and suggest that the open helix structure of the replication 'bubble' directs pol alpha-primase to initiate RNA primer synthesis near the ssDNA-dsDNA junction.

Cdk-interacting protein 1 directly binds with proliferating cell nuclear antigen and inhibits DNA replication catalyzed by the DNA polymerase delta holoenzyme.

Cdk-interacting protein 1 (Cip1) is a p53-regulated 21-kDa protein that inhibits several members of the cyclin-dependent kinase (CDK) family. It was initially observed in complexes containing CDK4, cyclin D, and proliferating cell nuclear antigen (PCNA). PCNA, in conjunction with activator 1, acts as a processivity factor for eukaryotic DNA polymerase (pol) delta, and these three proteins constitute the pol delta holoenzyme. In this report, we demonstrate that Cip1 can also directly inhibit DNA synthesis in vitro by binding to PCNA. Cip1 efficiently inhibits simian virus 40 replication dependent upon pol alpha, activator 1, PCNA, and pol delta, and this inhibition can be overcome by additional PCNA. Simian virus 40 DNA replication, catalyzed solely by high levels of pol alpha-primase complex, is unaffected by Cip1. Using the surface plasmon resonance technique, a direct physical interaction of PCNA and Cip1 was detected. We have observed that Cip1 efficiently inhibits synthesis of long (7.2 kb) but not short (10 nt) templates, suggesting that its association with PCNA is likely to impair the processive movement of pol delta during DNA chain elongation, as opposed to blocking assembly of the pol delta holoenzyme. The implications of the Cip1-PCNA interaction with respect to regulation of DNA synthesis, cell cycle checkpoint control, and DNA repair are discussed.

Replication of bovine papillomavirus type 1 origin-containing DNA in crude extracts and with purified proteins.

The in vitro replication of DNA containing the bovine papillomavirus (BPV-1) origin has been carried out with cell-free extracts from mouse FM3A and human HeLa cells. DNA synthesis required the E1 protein, the minimal origin of replication (nucleotides 7911-22 of the BPV-1 genome), and, at low levels of FM3A extract, the addition of the human single-stranded DNA-binding protein (also called RP-A or RF-A). The E2 protein was not absolutely required, but could stimulate DNA synthesis at low levels of E1. DNA synthesis was also reconstituted using purified proteins from HeLa cells. These protein factors included human single-stranded DNA-binding protein, topoisomerase I, and DNA polymerase (pol) alpha-primase complex. At low concentrations of pol alpha-primase complex, the formation of high molecular weight products was dependent on the addition of DNA polymerase delta holoenzyme containing proliferating cell nuclear antigen and activator 1, also called RF-C. We have overexpressed and isolated the E1 protein from bacteria. This protein also supported BPV DNA synthesis, both in crude extracts and with purified proteins suggesting that E1 phosphorylation is not required for BPV DNA replication in vitro.

Functional interactions between SV40 T antigen and other replication proteins at the replication fork

The functional interaction of simian virus 40 (SV40) large tumor antigen (T antigen) with DNA polymerase alpha (pol alpha)-primase complex, human single-stranded DNA binding protein (HSSB), and DNA polymerase delta (pol delta) holoenzyme, which includes pol delta, activator I (also called replication factor C), and proliferating cell nuclear antigen, at the replication fork was examined using the purified components that support SV40 DNA replication. Dilution of reaction mixtures during RNA primer synthesis revealed that T antigen remained associated continuously with the fork, while the pol alpha-primase complex dissociated from the complex during oligoribonucleotide synthesis. T antigen unwound duplex DNA from the SV40 core origin at a rate of 200 base pairs/min. Pol alpha-primase complex inhibited the rate of the unwinding reaction, and HSSB, pol alpha, and primase were all required for this effect. These requirements are the same as those essential for DNA primase-catalyzed oligoribonucleotide synthesis (Matsumoto, T., Eki, T., and Hurwitz, J. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 9712-9716). This result suggests that the pol alpha-primase complex interacts with T antigen and HSSB during the unwinding reaction to synthesize RNA primers and that the interaction decreases the rate of T antigen movement. While pol delta holoenzyme can elongate primed DNA chains at a rate of 400-600 nucleotides/min on singly primed phi X174 DNA, the rate of the leading strand synthesis catalyzed by pol delta holoenzyme in the SV40 replication system in vitro was about 200 nucleotides/min. This rate was similar to the unwinding rate catalyzed by T antigen. Thus, the rate of leading strand synthesis catalyzed by pol delta holoenzyme in vitro appears to be limited by the unwinding reaction catalyzed by T antigen.

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.

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).

Role of DNA polymerase alpha and DNA primase in simian virus 40 DNA replication in vitro.

The role of DNA polymerase alpha (pol alpha) and DNA primase has been investigated in the simian virus 40 (SV40) DNA replication system in vitro. Removal of pol alpha and primase activities from crude extracts of HeLa cells or monkey cells by use of an anti-pol alpha immunoaffinity column resulted in the loss of replication activity. The addition of purified pol alpha-primase complex isolated from HeLa cells or monkey cells restored the replication activity of depleted extracts. In contrast, the pol alpha-primase complex isolated from either mouse cells or calf thumus did not. Extracts prepared from mouse cells (a source that does not support replication of SV40) did not replicate SV40 DNA. However, the addition of purified pol alpha-primase complex isolated from HeLa cells activated mouse cell extracts. pol alpha and primase from HeLa cells were extensively purified and separated by a one-step immunoaffinity adsorption and elution procedure. Both activities were required to restore DNA synthesis; the addition of pol alpha or primase alone supported replication poorly. Crude extracts of HeLa cells that were active in SV40 replication catalyzed the synthesis of full-length linear double-stranded (RFIII) DNA in reaction mixtures containing poly(dT)-tailed pBR322 RFIII. Maximal activity was dependent on the addition of oligo(dA), ATP, and creatine phosphate and was totally inhibited by aphidicolin. Since pol alpha alone could not replicate this substrate and since there was no degradation of input DNA, we propose that other enzymatic activities associate with pol alpha, displace the non-template strand, and allow the enzyme to replicate through duplex regions.

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.