DNA primase associated with 10 S DNA polymerase α from calf thymus

Among multiple subspecies of DNA polymerase α of calf thymus, only 10 S DNA polymerase α had a capacity to initiate DNA synthesis on an unprimed single-stranded, circular M13 phage DNA in the presence of ribonucleoside triphosphates (DNA primase activity). The primase was copurified with 10 S DNA polymerase α through the purification and both activities cosedimented at 10 S through gradients of either sucrose or glycerol. Furthermore, these two activities were immunoprecipitated at a similar efficiency by a monoclonal antibody directed against calf thymus DNA polymerase α. These results indicate that the primase is tightly bound to 10 S DNA polymerase α. The RNA polymerizing activity was resistant to α-amanitin, required high concentration of all four ribonucleoside triphosphates (800 μM) for its maximal activity, and produced the limited length of oligonucleotides (around 10 nucleotides long) which were necessary to serve as a primer for DNA synthesis. Covalent bonding to RNA to DNA was strongly suggested by the nearest neighbour frequency analysis and the DNAase treatment. The DNA synthesis primed by the RNA oligomers may be carried out by the associating DNA polymerase α because it was strongly inhibited by araCTP, resistant to d₂TTP, and was also inhibited by aphidicolin but at relatively high concentration. The primase preferred single-stranded DNA as a template, but it also showed an activity on the double-stranded DNA from calf thymus at an efficiency of approx. 10% of that with single-stranded DNA.     

Replication of single-stranded porcine circovirus DNA by DNA polymerases α and δ

Porcine circovirus is the only mammalian DNA virus so far known to contain a single-stranded circular genome (Tischer et al. (1982) Nature 295, 64–66). Replication of its small viral DNA (1.76 kb) appears to be dependent on cellular enzymes expressed during S-phase of the cell cycle (Tischer et al. (1987) Arch. Virol. 96, 39–57). In this paper we have exploited the porcine circovirus genome to probe for in vitro initiation and elongation of DNA replication by different preparations of calf thymus DNA polymerase α and δ as well as by a partially purified preparation from pig thymus. The results indicated that three different purification fractions of calf thymus DNA polymerase α and one from pig thymus initiate DNA synthesis at several sites on the porcine circovirus DNA. It appears that the sites at which DNA primase synthesizes primers are not entirely random. Subsequent DNA elongation by a highly purified DNA polymerase α holoenzyme which had been isolated by the criterion of replicating single-stranded M13 DNA (Ottiger et al. (1987) Nucleic Acids Res. 15, 4789–4807) is very efficient. Complete conversion to the double-stranded form is obtained in less than 1 min. When the DNA synthesis by DNA polymerase α is blocked with the DNA polymerase α specific monoclonal antibody SJK 132-20 after initiation by DNA primase, DNA polymerase δ can efficiently replicate from the primers. This in vitro DNA replication system may be used in analogy to the bacteriophage systems in E. coli to study initiation and elongation of DNA replication.     

Structural study of immunoaffinity-purified DNA polymerase α-DNA primase complex from calf thymus

The DNA polymerase α-DNA primase complex was purified over 17 000-fold to near homogeneity from calf thymus using an immunoaffinity column. Sodium dodecyl sulfate gel electrophoresis revealed three polypeptides with molecular weights of 140, 50 and 47 kDa, in a ratio of 1:2:0.25. The complex showed a sedimentation coefficient of 9.7 S, a Stokes radius of 56 Å and a native molecular weight of 250–260 kDa. Taken together, the data suggest that the calf thymus dNA polymerase α-DNA primase complex is essentially a heterotrimer of large (140 kDa) and small (50 kDa) subunits in a ratio of 1:2, with a globular conformation. Electron-microscopic studies of the complex revealed a spherical particle of 120 Å in diameter, in agreement with the physicochemical results. The binding of the complex to DNA was also demonstrated.     

Purification and properties of a DNA primase from Nicotianatabacum

A DNA primase was isolated from a nuclear fraction from leaves of tobacco (Nicotiana tabacum L. cv. Samsun) and from purified nuclei prepared from tobacco suspension culture cells. The DNA primase was purified to homogeneity (i) for preparations from leaves, by ammonium sulphate fractionation, followed by chromatography on columns of phosphocellulose, Q-Sepharose, heparin-Sepharose and single-stranded DNA cellulose, and sedimentation in a glycerol gradient, or (ii) for preparations from cells, by chromatography on single-stranded DNA cellulose, followed by ammonium sulphate precipitation and chromatography on columns of High Q, heparin-Sepharose and Mono Q. In glycerol gradients, the DNA primase sedimented at a rate corresponding to a molecular mass of about 120 kDa. In SDS-polyacrylamide gel electrophoresis, the primase was resolved into two polypeptide subunits of 63 kDa and 53 kDa, which are similar in size to the primase subunits of animal and yeast DNA polymerase α-primase complexes. On poly(dT) or phage M13 single-stranded DNA templates, the DNA primase catalysed the synthesis of oligoribonucleotides up to 20 nucleotides in length, which could serve as primers for DNA synthesis catalysed by Escherichia coli DNA polymerase. Primase activity was dependent on a template, magnesium ions and ATP; it was resistant to aphidicolin and rifampicin, but was strongly inhibited by N-ethylmaleimide. This is the first report of the purification to homogeneity of a plant DNA primase. Received: 8 May 1997 / Accepted: 5 June 1997     

DNA primase activity from wheat embryos

DNA primase is a recently discovered enzyme capable of synthesizing short primers involved in the initiation of DNA replication.Partially purified preparations from 4 h germinated wheat embryos or commercial wheat germ are able to catalyze the ribonucleoside triphosphate dependent synthesis of DNA with poly dT and M₁₃ single stranded DNA as templates. DNA synthesis is completely dependent on the presence of template and primase. Primase activity from wheat embryos has a molecular weight of about 110000 and a sedimentation coefficient of 5S. The enzyme activity is not inhibited by -amanitin (1 mg/ml) or aphidicolin when the latter is assayed with endogeneous plant DNA polymerase activity. Alkaline hydrolysis of the product synthesized in the presence of [-³²P]dATP and poly dT generates [³²P]-labeled 3(2)AMP showing that a ribo-deoxynucleotide linkage is formed. The size of the oligoribonucleotide primer varies from 2 to 15 residues. Most of the wheat DNA polymerase activity can be eliminated by phosphocellulose chromatography, since the bulk of plant DNA primase is not retained by this resin. Nevertheless, a small but significant amoung of DNA polymerase activity is found associated with DNA primase. By using different inhibitors of DNA polymerase different templates, we have found good indications that DNA polymerase A (-like) is associated with the DNA primase. Moreover, when the previously purified DNA polymerases from wheat embryos (2) were assayed in the presence of primase activity, only DNA polymerase A was able to stimulate DNA synthesis.     

No discrete complexes containing DNA polymerase α activity can be solubilized from the heat-stabilized nuclear matrix prepared from HeLa S3 cells

Most of the DNA polymerase α activity, bound to the heat-stabilized nuclear matrix prepared from HeLa S3 cells, was released as a matrix extract by sonication. When the extract was centrifuged in a 5–20 per cent linear sucrose gradient no definite peaks of activity could be identified. Most of the activity sedimented to the bottom of the tube under all the conditions tested, whilst the remaining activity was associated with matrix fragments of various and irregular size. No 10 S complexes, containing polymerase activity, were seen after incubation of the extract for 16 h before centrifugation. Other solubilization procedures (i.e. treatment of the matrix with chelating agents, high pH associated with reducing agents, ionic and nonionic detergents) failed to produce release of matrix-bound DNA polymerase α activity. In contrast, we released 10 S complexes, containing polymerase activity, from the matrix prepared from nuclei not exposed to heat. We conclude that a 37°C incubation of isolated nuclei before extraction with 2 M NaCl and DNase I digestion causes DNA polymerase α to bind to the nuclear matrix in a form that cannot subsequently be released as discrete components, at variance with previous results obtained with the matrix prepared from regenerating rat liver.     

DNA repair in ultraviolet-irradiated HeLa cells is disrupted by aphidicolin: The inhibition of repair need not imply the absence of repair synthesis

Aphidicolin, a potent and specific inhibitor of eukaryotic DNA polymerase α, has been reported to inhibit repair DNA synthesis in ultraviolet-irradiated, normal human fibroblasts but not in HeLa cells. By the use of assays for repair other than the measurement of repair synthesis, it is shown here that repair in HeLa cells is in fact susceptible to aphidicolin. Severe inhibition of DNA repair, with failure of individual repair events to be completed, and a smaller number of lesions removed, can occur even though repair synthesis continues.     

Structural basis for the interaction of SARS‐CoV‐2 virulence factor nsp1 with DNA polymerase α–primase

The molecular mechanisms that drive the infection by the severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2)—the causative agent of coronavirus disease 2019 (COVID‐19)—are under intense current scrutiny to understand how the virus operates and to uncover ways in which the disease can be prevented or alleviated. Recent proteomic screens of the interactions between viral and host proteins have identified the human proteins targeted by SARS‐CoV‐2. The DNA polymerase α (Pol α)–primase complex or primosome—responsible for initiating DNA synthesis during genomic duplication—was identified as a target of nonstructural protein 1 (nsp1), a major virulence factor in the SARS‐CoV‐2 infection. Here, we validate the published reports of the interaction of nsp1 with the primosome by demonstrating direct binding with purified recombinant components and providing a biochemical characterization of their interaction. Furthermore, we provide a structural basis for the interaction by elucidating the cryo‐electron microscopy structure of nsp1 bound to the primosome. Our findings provide biochemical evidence for the reported targeting of Pol α by the virulence factor nsp1 and suggest that SARS‐CoV‐2 interferes with Pol α''s putative role in the immune response during the viral infection.     

真核生物引发酶及其作用

在真核生物中,几乎所有新DNA链的起始都利用引发酶来合成长约8~12个核苷酸的RNA作为引物。目前所知的所有真核生物的DNA引发酶均由大小约为58×103和49×103的两个亚基(p58和p49)组成,这两个亚基与DNA聚合酶α形成紧密的复合物。对引发酶中的活性中心及其反应机制均有相当的了解。引发酶不仅在DNA得复制中起着重要作用,而且在复制偶连的DNA修复及端粒的保持中起着关键性的作用,因而对染色体的稳定起着重要作用。     

Catalytic properties of DNA polymerase α activity associated with the heat-stabilized nuclear matrix prepared from HeLa S3 cells

We have investigated whether or not ATP or other nucleoside di- and trisphosphates (including some nonhydrolysable ATP analogues) can stimulate the activity and/or the processivity of DNA polymerase α associated with the nuclear matrix obtained from HeLa S3 cell nuclei that had been stabilized at 37°C prior to subfractionation, as has been reported previously for DNA polymerase α bound to the nuclear matrix prepared from 22-h regenerating rat liver. We have found that HeLa cell matrix-associated DNA polymerase α activity could not be stimulated at all by ATP or other nucleotides, a behaviour which was shared also by DNA polymerase α activity that solubilizes from cells during the isolation of nuclei and that is thought to be a form of the enzyme not actively engaged in DNA replication. Moreover, the processivity of matrix-bound DNA polymerase α activity was low (< 10 nucleotides). These results were obtained with the matrix prepared with either 2M NaCl or 0·25 M (NH₄)₂SO₄ and led us to consider that a 37° incubation of isolated nuclei renders resistant to high-salt extraction a form of DNA polymerase α which is unlikely to be involved in DNA replication in vivo.     

Prereplicative increase of nuclear matrix-bound DNA polymerase-α and primase activities in HeLa S3 cells following dilution of long-term cultures

We investigated the association of DNA polymerase and DNA primase activity with the nuclear matrix in HeLa S3 cells diluted with fresh medium after having been cultured without any medium change for 7 days. Flow cytometric analysis demonstrated that just before dilution about 85% of the cells were in the G₁ phase of the cycle, whereas 8% were in the S phase. After dilution with fresh medium, 18–22 h were required for the cell population to attain a stable distribution with respect to the cell cycle. At that time, about 38% of the cells were in the S phase. DNA polymerase and DNA primase activity associated with the nuclear matrix prepared from cells just before dilution represented about 10% of nuclear activity. As judged by [³H]-thymidine incorporation and flow cytometric analysis, an increase in the number of S-phase cells was evident at least 6 h after dilution. However, as early as 2 h after dilution into fresh medium, a striking prereplicative increase of the two activitites was seen in the nuclear matrix fraction but not in cytosol or isolated nuclei. Both DNA polymerase and primase activities bound to the matrix were about 60% of nuclear activity. Overall, the nuclear matrix was the cell fraction where the highest induction (about 10-fold) of both enzymatic activities was seen at 30 h after dilution, whereas in cytosol and isolated nuclei the increase was about two- and fourfold, respectively. Typical immunofluorescent patterns given by an antibody to 5-bromodeoxyuridine were seen after dilution. These findings, which are at variance with our own previous results obtained with cell cultures synchronized by either a double thymidine block or aphidicolin exposure, strengthen the contention that DNA replication is associated with an underlying nuclear structure and demonstrate the artifacts that may be generated by procedures commonly used to synchronize cell cultures. J. Cell. Biochem. 71:11–20, 1998. © 1998 Wiley-Liss, Inc.     

DNA polymerase κ‐dependent DNA synthesis at stalled replication forks is important for CHK1 activation

Formation of primed single‐stranded DNA at stalled replication forks triggers activation of the replication checkpoint signalling cascade resulting in the ATR‐mediated phosphorylation of the Chk1 protein kinase, thus preventing genomic instability. By using siRNA‐mediated depletion in human cells and immunodepletion and reconstitution experiments in Xenopus egg extracts, we report that the Y‐family translesion (TLS) DNA polymerase kappa (Pol κ) contributes to the replication checkpoint response and is required for recovery after replication stress. We found that Pol κ is implicated in the synthesis of short DNA intermediates at stalled forks, facilitating the recruitment of the 9‐1‐1 checkpoint clamp. Furthermore, we show that Pol κ interacts with the Rad9 subunit of the 9‐1‐1 complex. Finally, we show that this novel checkpoint function of Pol κ is required for the maintenance of genomic stability and cell proliferation in unstressed human cells.     

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination

We have previously purified and characterized wheat germ DNA polymerases A and B. To determine the role played by DNA polymerases A and B in DNA replication, we have measured the level of their activities during wheat embryo germination. The level of cellular proteins known to be associated with DNA synthesis such as PCNA and DNA primase were also investigated. The activity of DNA polymerase A gradually increased reaching a maximal level at 12 h after germination. Three days later, only a residual activity was detected. DNA polymerase B showed the same pattern during germination with very similar changes in activity. Our results indicate a striking correlation between maximal activities of DNA polymerase A, DNA polymerase B and optimal levels of DNA synthesis. These results support a replicative role of these enzymes. The activity of wheat DNA primase that copurifies with DNA polymerase A also increases during wheat germination. Taking together all its properties, and in spite of its behaviour with some inhibitors, DNA polymerase A may be considered as the plant counterpart of animal DNA polymerase . Concerning DNA polymerase B we have previously shown that PCNA stimulates its processivity. Besides studying the changes of DNA polymerases A and B and DNA primase we have also studied changes in PCNA during germination. We show that PCNA is present in wheat embryos at a constant relatively high level during the first 24 h of germination. After 48 h, the absence of PCNA is concomitant with an important decrease in DNA polymerase B activity. In this report we confirm the behaviour of DNA polymerase B as a -like activity.Département de Biologie, Université de Drah-Lmraz,Fez, Maroc     

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.     

p53 suppression overwhelms DNA polymerase eta deficiency in determining the cellular UV DNA damage response

Xeroderma pigmentosum variant (XP-V) cells lack the damage-specific DNA polymerase eta and have normal excision repair but show defective DNA replication after UV irradiation. Previous studies using cells transformed with SV40 or HPV16 (E6/E7) suggested that the S-phase response to UV damage is altered in XP-V cells with non-functional p53. To investigate the role of p53 directly we targeted p53 in normal and XP-V fibroblasts using short hairpin RNA. The shRNA reduced expression of p53, and the downstream cell cycle effector p21, in control and UV irradiated cells. Cells accumulated in late S phase after UV, but after down-regulation of p53 they accumulated earlier in S. Cells in which p53 was inhibited showed ongoing genomic instability at the replication fork. Cells exhibited high levels of UV induced S-phase gammaH2Ax phosphorylation representative of exposed single strand regions of DNA and foci of Mre11/Rad50/Nbs1 representative of double strand breaks. Cells also showed increased variability of genomic copy numbers after long-term inhibition of p53. Inhibition of p53 expression dominated the DNA damage response. Comparison with earlier results indicates that in virally transformed cells cellular targets other than p53 play important roles in the UV DNA damage response.     

Comparison among DNA polymerases 1, 2 and 3 from maize embryo axes. A DNA primase activity copurifies with DNA polymerase 2

Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strenghtening the suggestion that polymerase 2 is a replicative enzyme, of the -type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature.     

MiR‐149 sensitizes esophageal cancer cell lines to cisplatin by targeting DNA polymerase β

Human DNA polymerase β (polβ) is a small, monomeric protein essential for short‐patch base excision repair (BER). polβ plays an important role in the regulation of chemotherapy sensitivity in tumour cells. In this study, we determined that the expression levels of polβ mRNA and miR‐149 in tumour tissues were significantly higher than in adjacent non‐tumour tissues. We also found that the expression level of miR‐149 in EC tumour tissues was inverse to that of polβ expression. Bioinformatics analysis and dual‐luciferase reporter assay predicted that miR‐149 negatively regulates polβ expression by directly binding to its 3′UTR. CCK‐8 assay indicated that miR‐149 could enhance the anti‐proliferative effects of cisplatin in EC1 and EC9706 cell lines. Flow cytometry, caspase 3/7 activity, and immunofluorescence microscopy results indicated that miR‐149 could enhance the apoptotic effects of cisplatin in EC1 and EC9706 cell lines. We also showed that the expression of polβ lacking the 3′UTR sequence could override the proliferative and apoptotic functions of miR‐149, suggesting that miR‐149 negatively regulates polβ expression by binding to its 3′UTR. Surface plasmon resonance results also showed that miR‐149 could bind with wild‐type polβ. In addition, we identified a new variant of polβ (C1134G). In conclusion, this study confirms that miR‐149 may enhance the sensitivity of EC cell lines to cisplatin by targeting polβ, and that miR‐149 may be unable to regulate the C1134G variant of polβ. Based on these findings, potential drugs could be developed with a focus on enhanced sensitivity of EC patients to chemotherapy.     

Mechanism of Bidirectional Leading-Strand Synthesis Establishment at Eukaryotic DNA Replication Origins

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