Exonuclease activities associated with DNA polymerases alpha and beta of the archaebacterium Halobacterium halobium.

alpha-like and beta-like DNA polymerases have previously been isolated from a halophilic archaebacterium Halobacterium halobium. In this report, we show that the alpha-like DNA polymerase has an associated 3' to 5'-exonuclease activity which is specific for single-stranded DNA, sensitive to both aphidicolin and N-ethylmaleimide and dependent on high salt concentrations like the polymerase activity. As this DNA polymerase has been shown to contain a primase activity, it may be considered as the equivalent to both eukaryotic DNA polymerases alpha and delta. As shown by glycerol-gradient centrifugation and electrophoresis under denaturing conditions, the beta-like polymerase would appear to have a monomeric structure and comprise of a single 65-kDa polypeptide. This DNA polymerase has both 3' to 5'-exonuclease and 5' to 3'-exonuclease activities which, contrary to polymerase activity, are inhibited by high salt concentrations.     

Characterization of a stable, major DNA polymerase alpha species devoid of DNA primase activity.

We have purified from Xenopus laevis ovaries a major DNA polymerase alpha species that lacked DNA primase activity. This primase-devoid DNA polymerase alpha species exhibited the same sensitivity as the DNA polymerase DNA primase alpha to BuAdATP and BuPdGTP, nucleotide analogs capable of distinguishing between DNA polymerase delta and DNA polymerase DNA primase alpha. The primase-devoid DNA polymerase alpha species also lacked significant nuclease activity indicative of the alpha-like (rather than delta-like) nature of the DNA polymerase. Using a poly(dT) template, the primase-devoid DNA polymerase alpha species elongated an oligo(rA10) primer up to 51-fold more effectively than an oligo(dA10) primer. In direct contrast, the DNA polymerase DNA primase alpha complex showed only a 4.6-fold preference for oligoribonucleotide primers at the same template/primer ratio. The catalytic differences between the two DNA polymerase alpha species were most dramatic at a template/primer ratio of 300. The primase-devoid DNA polymerase alpha species was found at high levels throughout oocyte and embryonic development. This suggests that the primase-devoid DNA polymerase alpha species could play a physiological role during DNA chain elongation in vivo, even if it is chemically related to DNA polymerase DNA primase alpha.     

Polypeptide structure of DNA primase from a yeast DNA polymerase-primase complex

An immunoaffinity chromatographic procedure was developed to purify DNA polymerase-DNA primase complex from crude soluble extracts of yeast cells. The immunoabsorbent column is made of mouse monoclonal antibody to yeast DNA polymerase I covalently linked to Protein A-Sepharose. Purification of the complex involves binding of the complex to the immunoabsorbent column and elution with concentrated MgCl2 solutions. After rebinding to the monoclonal antibody column free primase activity is selectively eluted with a lower concentration of MgCl2. Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed the presence of five major peptides, p180, p140, p74, p58, and p48 in the immunoaffinity-purified DNA polymerase-DNA primase complex. Free primase and free polymerase fractions obtained by fractionation on the immunoabsorbent column were analyzed on activity gels and immunoblots. These analyses showed that p180 and p140 are DNA polymerase peptides. Two polypeptides of 58 and 48 kDa co-fractionated with the free yeast DNA primase. From sucrose gradient analysis we estimate a molecular weight of 110 kDa for the native DNA primase.     

Pea chloroplast DNA primase: characterization and role in initiation of replication

A DNA primase activity was isolated from pea chloroplasts and examined for its role in replication. The DNA primase activity was separated from the majority of the chloroplast RNA polymerase activity by linear salt gradient elution from a DEAE-cellulose column, and the two enzyme activities were separately purified through heparin-Sepharose columns. The primase activity was not inhibited by tagetitoxin, a specific inhibitor of chloroplast RNA polymerase, or by polyclonal antibodies prepared against purified pea chloroplast RNA polymerase, while the RNA polymerase activity was inhibited completely by either tagetitoxin or the polyclonal antibodies. The DNA primase activity was capable of priming DNA replication on single-stranded templates including poly(dT), poly(dC), M13mp19, and M13mp19_+ 2.1, which contains the AT-rich pea chloroplast origin of replication. The RNA polymerase fraction was incapable of supporting incorporation of ³H-TTP in in vitro replication reactions using any of these single-stranded DNA templates. Glycerol gradient analysis indicated that the pea chloroplast DNA primase (115–120 kDa) separated from the pea chloroplast DNA polymerase (90 kDa), but is much smaller than chloroplast RNA polymerase. Because of these differences in size, template specificity, sensitivity to inhibitors, and elution characteristics, it is clear that the pea chloroplast DNA primase is an distinct enzyme form RNA polymerase. In vitro replication activity using the DNA primase fraction required all four rNTPs for optimum activity. The chloroplast DNA primase was capable of priming DNA replication activity on any single-stranded M13 template, but shows a strong preference for M13mp19+2.1. Primers synthesized using M13mp19+2.1 are resistant to DNase I, and range in size from 4 to about 60 nucleotides.     

Purification and properties of an accessory protein for DNA polymerase alpha/primase

A protein that stimulates DNA polymerase alpha/primase many-fold on unprimed poly(dT) was purified to homogeneity from extracts of cultured mouse cells. The protein contains polypeptides of approximately 132 and 44 kDa, and the total molecular mass of 150 kDa calculated from Stokes radius (54 A) and sedimentation coefficient (6.7 S) indicates that it contains one each of the two subunits. The purified "alpha accessory factor" (AAF) also stimulates DNA polymerase alpha/primase in the self-primed reaction with unprimed single-stranded DNA. In addition to these effects on the coordinate activities of DNA polymerase alpha and DNA primase, stimulatory effects were also demonstrated separately on both the polymerase and primase activities of the enzyme complex. However, there was no stimulation with DNase-treated ("activated") DNA under normal conditions for assay of DNA polymerase alpha. The stimulatory activity of mouse AAF is highly specific for DNA polymerase alpha/primase; no effect was observed with mouse DNA polymerases beta, gamma, or delta, nor with retroviral, bacteriophage, or bacterial DNA polymerases. Mouse AAF stimulated human DNA polymerase alpha/primase with several different templates, similar to results with the mouse enzyme. However, it had very little effect on the DNA polymerase/primase from either Drosophila embryo or from yeast.     

An alpha-like DNA polymerase from Halobacterium halobium

Two DNA polymerases have been isolated from extracts of Halobacterium halobium, one having a sedimentation coefficient of 11 S, designated as alpha-like polymerase and possessing the following characteristics. It is sensitive to both aphidicolin and N-ethylmaleimide but indifferent to the presence of a dideoxynucleoside triphosphate. Therefore this polymerase is very similar to the alpha DNA polymerase of eukaryotes. The enzyme requires 5 M NaCl for maximum activity. The other polymerase has a sedimentation coefficient of 4.4 S and is resistant to both aphidicolin and N-ethylmaleimide. However, it is inhibited by a dideoxynucleoside triphosphate.     

Photoaffinity labeling of DNA polymerase alpha DNA primase complex based on the catalytic competence of a dNTP reactive analog.

FABdCTP was found to be a substrate of DNA polymerization catalyzed by a DNA polymerase alpha-DNA primase complex on the 5'-GTGAGTAAGTGGAGTTTGGCACGAT-3' template and 3'-CTCAAACCGT-5' primer. After complete primer extension in the presence of FABdCTP under UV-irradiation of the reaction mixture, 70% of the template was covalently linked to the primer. Labeling of the 165 kDa subunit of the DNA polymerase alpha, 59 kDa and 49 kDa subunits of the DNA primase and an unknown protein with apparent molecular weight of 31 kDa was observed. By another way of protein labeling FABdCTP was covalently bound to the subunits of the enzyme under UV irradiation and then this moiety was introduced into the 3'-end of the 5'-[32P]primer by the catalytic activity of DNA polymerase or DNA primase. In this case covalent labeling of the 165 kDa, 49 kDa and 31 kDa subunits was observed.     

Immunochemical detection of a primase activity related subunit of DNA polymerase alpha from human and mouse cells using the monoclonal antibody

A hybrid cell line (HDR-854-E4) secreting monoclonal antibody (E4 antibody) against a subunit of human DNA polymerase alpha was established by immunizing mice with DNA replicase complex (DNA polymerase alpha-primase complex) prepared from HeLa cells. The E4 antibody immunoprecipitates DNA replicase complex from both human and mouse cells. The E4 antibody neutralizes the primase activity as assessed either by the direct primase assay (incorporation of [alpha-32P]AMP) or by assay of DNA polymerase activity coupled with the primase activity using unprimed poly(dT) as a template. The E4 antibody does not neutralize DNA polymerase alpha activity with the activated calf thymus DNA as a template. Western immunoblotting analysis shows that the E4 antibody binds to a polypeptide of 77 kilodaltons (kDa) which is tightly associated with DNA polymerase alpha. The 77-kDa polypeptide was distinguished from the catalytic subunit (160 and 180 kDa) for DNA synthesis which was detected by another monoclonal antibody, HDR-863-A5. Furthermore, it is unlikely that the 77-kDa peptide is the primase, since we found that the E4 antibody also immunoprecipitates the mouse 7.3S DNA polymerase alpha which has no primase activity, and Western immunoblotting analysis shows that the 77-kDa polypeptide is a subunit of the 7.3S DNA polymerase alpha. Furthermore, after dissociation of the primase from mouse DNA replicase by chromatography on a hydroxyapatite column in the presence of dimethyl sulfoxide and ethylene glycol, the 77-kDa polypeptide is associated with DNA polymerase alpha, and not with the primase.(ABSTRACT TRUNCATED AT 250 WORDS)     

DNA polymerase alpha-DNA primase from human placenta. Immunoaffinity purification and preliminary characterization

Highly purified DNA polymerase alpha-DNA primase from normal human tissue (human placenta) has been prepared by immunoaffinity purification on immobilized anti-human DNA polymerase alpha monoclonal antibody SJK 287-38. According to data from SDS electrophoresis this preparation consists of subunits of 180, 160, 145, 140 kDa (a cluster of DNA-polymerizing subunits), 73 kDa (function unknown) and 59, 52 kDa (corresponding to primase). Three active enzyme forms of 270, 460 and 575 kDa have been revealed using native electrophoresis followed by detection of DNA polymerase activity.     

Eukaryotic DNA primase appears to act as oligomer in DNA-polymerase-alpha--primase complex.

Human placenta and calf thymus DNA-polymerase-alpha-primases were analyzed using native gradient-polyacrylamide-gel electrophoresis followed by overlay assays of polymerase and primase activities. The human enzyme contained three catalytically active native forms of 330, 440 and 560 kDa and the bovine enzyme five forms of 330, 440, 500, 590 and 660 kDa. Of the various DNA polymerase forms, only the largest (560 kDa for human DNA polymerase and 590 kDa and 660 kDa for bovine DNA polymerase) contained primase activity. Titration of human DNA-polymerase-alpha-primase with DNA-polymerase-free primase caused the conversion of the 440-kDa to the 560-kDa form. The data favour the idea that primase binds to DNA polymerase alpha as an oligomer of 3 primases/polymerase core. In addition, the ability of primase to utilize oligoriboadenylates containing (prA)n or pp(prA)n was investigated. The primase elongated pp(prA)2-7 up to nanoadenylates or decaadenylates, but did not add 9 or 10 mononucleotides to a preexistent primer. In contrast to pp(prA)n less than 10, (prA)n less than 10 were rather poor primers for the primase. Both pp(prA)8,9 and (prA)n greater than 10 were elongated by primase, producing characteristic multimeric oligonucleotides. The possible connection of the structure of the DNA-polymerase-alpha-primase complex with the catalytical properties of primase is discussed.     

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 isolated from the yeast DNA primase-DNA polymerase complex. Immunoaffinity purification and analysis of RNA primer synthesis

We have utilized immunoaffinity chromatography as a means of efficiently isolating a stable yeast DNA primase from the DNA primase-DNA polymerase complex, allowing identification of the polypeptides associated with this DNA primase activity and comparison of its enzymatic properties with those of the larger protein complex. A mouse monoclonal antibody specifically recognizing the DNA polymerase subunit was used to purify the complex. Stable DNA primase was subsequently separated from the complex in high yield. The highly purified protein fraction which bound to the DNA polymerase antibody column consisted of polypeptides with apparent molecular masses of 180, 86, 70, 58, 49, and 47 kDa. DNA primase activity eluted with a fraction containing only the 58-, 49-, and 47-kDa polypeptides. Partial chemical cleavage analysis of these three proteins demonstrated that the 49- and 47-kDa polypeptides are structurally related while the 58-kDa protein is unrelated to the other two. A DNA primase inhibitory monoclonal antibody was able to inhibit the activity of the purified DNA primase as well as the activity of the enzyme in the larger complex. In immunoprecipitation experiments, all three polypeptides were found in the immune complex. Thus, these three polypeptides are sufficient for DNA primase activity. In reactions using ribonucleotide substrates and natural as well as synthetic DNA templates, the purified DNA primase exhibited the same precise synthesis of unit length oligomers as did the larger protein complex and was able to extend these RNA oligomers by one additional unit length. An examination of the effects of deoxynucleotides on these DNA primase-catalyzed reactions revealed that the yeast DNA primase is an RNA-polymerizing enzyme and lacks significant DNA-polymerizing activity under the conditions tested.     

DNA polymerase I and DNA primase complex in yeast

Chromatographic analysis of poly(dT) replication activity in fresh yeast extracts showed that the activities required co-fractionate with the yeast DNA polymerase I. Since poly(dT) replication requires both a primase and a DNA polymerase, the results of the fractionation studies suggest that these two enzymes might exist as a complex in the yeast extract. Sucrose gradient analysis of concentrated purified yeast DNA polymerase I preparations demonstrates that the yeast DNA polymerase I does sediment as a complex with DNA primase activity. Two DNA polymerase I peptides estimated at 78,000 and 140,000 Da were found in the complex that were absent from the primase-free DNA polymerase fraction. Rabbit anti-yeast DNA polymerase I antibody inhibits DNA polymerase I but not DNA primase although rabbit antibodies are shown to remove DNA primase activity from solution by binding to the complex. Mouse monoclonal antibody to yeast DNA polymerase I binds to free yeast DNA polymerase I as well as the complex, but not to the free DNA primase activity. These results suggest that these two activities exist as a complex and reside on the different polypeptides. Replication of poly(dT) and single-stranded circular phage DNA by yeast DNA polymerase I and primase requires ATP and dNTPs. The size of the primer produced is 8 to 9 nucleotides in the presence of dNTPs and somewhat larger in the absence of dNTPs. Aphidicolin, an inhibitor of yeast DNA polymerase I, is not inhibitory to the yeast DNA primase activity. The primase activity is inhibited by adenosine 5'-(3-thio)tri-phosphate but not by alpha-amanitin. The association of yeast DNA polymerase I and yeast DNA primase can be demonstrated directly by isolation of the complex on a column containing yeast DNA polymerase I mouse monoclonal antibody covalently linked to Protein A-Sepharose. Both DNA polymerase I and DNA primase activities are retained by the column and can be eluted with 3.5 M MgCl2. Part of the primase activity can be dissociated from DNA polymerase on the column with 1 M MgCl2 and this free primase activity can be detected as poly(dT) replication activity in the presence of Escherichia coli polymerase I.     

Characterization of DNA primase separated from DNA polymerase alpha-DNA primase complex of calf thymus

It has been shown that DNA primase activity is tightly associated with 10S DNA polymerase alpha from calf thymus (Yoshida, S. et al. (1983) Biochim. Biophys. Acta 741, 348-357). In the present study, the primase activity was separated from DNA polymerase alpha by treating purified 10S DNA polymerase alpha with 3.4 M urea followed by a fast column chromatography (Pharmacia FPLC, Mono Q column equilibrated with 2 M urea). Ten to 20 % of the primase activity was separated from 10S DNA polymerase alpha by this procedure but 80-90% remained in the complex. The separated primase activity sedimented at 5.6S through a gradient of glycerol. The separated primase was strongly inhibited by araATP (Ki = 10 microM) and was also sensitive to salts such as KCl (50% inhibition at 30 mM). The primase used poly(dT) or poly(dC) as templates efficiently, but showed little activity with poly(dA) or poly(dI). These properties agree well with those of the primase activity in the DNA polymerase alpha-primase complex (10S DNA polymerase alpha). These results indicate that the calf thymus primase may be a part of the 10S DNA polymerase alpha and its enzymological characters are preserved after separation from the complex.     

Maize replicative alpha-type DNA polymerase: separation of polymerase and primase activities and recognition of primase subunits

DNA polymerase and DNA primase activities in the maize α-type DNA polymerase 2 were dissociated and DNA polymerase-free DNA primase was studied. DNA primase synthesized primers that were 8–34 nucleotides long, with more intense bands at 15–17 nucleotides in length. DNA polymerase 1 (a putative δ-type enzyme) or DNA polymerase 2 were assayed after template-priming with purified DNA primase and showed a differential use of templates: whereas DNA polymerase 2 used a polydT template more efficiently than a natural template, DNA polymerase 1 used both of them poorly. The molecular size of DNA primase was estimated to be 68 kDa by gel filtration, western blotting and by a DNA primase 'trapping' assay.     

A unique subpopulation of murine DNA polymerase alpha/primase specifically interacts with polyomavirus T antigen and stimulates DNA replication.

Murine cells or cell extracts support the replication of plasmids containing the replication origin (ori-DNA) of polyomavirus (Py) but not that of simian virus 40 (SV40), whereas human cells or cell extracts support the replication of SV40 ori-DNA but not that of Py ori-DNA. It was shown previously that fractions containing DNA polymerase alpha/primase from permissive cells allow viral ori-DNA replication to proceed in extracts of nonpermissive cells. To extend these observations, the binding of Py T antigen to both the permissive and nonpermissive DNA polymerase alpha/primase was examined. Py T antigen was retained by a murine DNA polymerase alpha/primase but not by a human DNA polymerase alpha/primase affinity column. Likewise, a Py T antigen affinity column retained DNA polymerase alpha/primase activity from murine cells but not from human cells. The murine fraction which bound to the Py T antigen column was able to stimulate Py ori-DNA replication in the nonpermissive extract. However, the DNA polymerase alpha/primase activity in this murine fraction constituted only a relatively small proportion (approximately 20 to 40%) of the total murine DNA polymerase alpha/primase that had been applied to the column. The DNA polymerase alpha/primase purified from the nonbound murine fraction, although far more replete in this activity, was incapable of supporting Py DNA replication. The two forms of murine DNA polymerase alpha/primase also differed in their interactions with Py T antigen. Our data thus demonstrate that there are two distinct populations of DNA polymerase alpha/primase in murine cells and that species-specific interactions between T antigen and DNA polymerases can be identified. They may also provide the basis for initiating a novel means of characterizing unique subpopulations of DNA polymerase alpha/primase.     

Immunological analysis of the polypeptide structure of calf thymus DNA polymerase-primase complex

Five major polypeptides are found in immunoaffinity-purified calf thymus DNA polymerase-DNA primase complex: 185, 160, 68, 55, and 48 kDa. Individual polypeptides purified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were used to produce antibodies in rabbits to aid in identifying the relationships between these polypeptides by immunoblotting and enzyme neutralization procedures. Immunoblot analyses showed that the 160-kDa peptide is derived from the 185-kDa peptide and the 48-kDa peptide is derived from the 68-kDa peptide while antibodies to the 55-kDa peptide do not cross-react with other peptides found in the complex. Direct enzyme neutralization studies demonstrated that antibodies to 185- and 160-kDa peptides inhibit DNA polymerase activity in the complex, confirming earlier suggestions that these peptides are the catalytic peptides for DNA polymerase. DNA primase activity in the complex is inhibited by antibodies to 68-, 55-, and 48-kDa peptides and to a lesser extent by antibodies to the 160-kDa peptide. Free DNA primase isolated from the complex was estimated to have a native molecular weight of about 110,000. The 55- and 48-kDa peptides are found to be associated with the free primase activity. Rabbit antibodies to both 55- and 48-kDa peptides are inhibitory to this primase activity. From these results we suggest that the native calf thymus DNA polymerase-DNA primase complex contains only three unique peptides with the 185-kDa peptide as the catalytic peptide of DNA polymerase and the 55- and 68-kDa peptides constituting the primase peptides. A model illustrating the roles of these peptides in initiation and replication of DNA is presented.     

Resolution and purification of free primase activity from the DNA primase-polymerase alpha complex of HeLa cells.

DNA primase activity has been resolved from a purified DNA primase-polymerase alpha complex of HeLa cells by hydrophobic affinity chromatography on phenylSepharose followed by chromatography on hexylagarose. This procedure provides a good yield (55%) of DNA primase that is free from polymerase alpha. The free DNA primase activity was purified to near homogeneity and its properties characterized. Sodium dodecyl sulfate polyacrylamide gel electrophoretic analysis of the purified free DNA primase showed a major protein staining band of Mr 70,000. The native enzyme in velocity sedimentation has an S20'W of 5. DNA primase synthesizes RNA oligomers with single-stranded M-13 DNA, poly(dT) and poly(dC) templates that are elongated by the DNA polymerase alpha in a manner that has already been described for several purified eukaryotic DNA primase-polymerase alpha complexes. The purified free DNA primase activity is resistant to neutralizing anti-human DNA polymerase alpha antibodies, BuPdGTP and aphidicolin that specifically inhibit the free DNA polymerase alpha and also DNA polymerase alpha complexed with the primase. The free primase activity is more sensitive to monovalent salt concentrations and is more labile than polymerase alpha. Taken together these results indicate that the DNA primase and polymerase alpha activities of the DNA primase-polymerase alpha complex reside on separate polypeptides that associate tightly through hydrophobic interactions.     

Identification and characterization of a DNA primase activity present in herpes simplex virus type 1-infected HeLa cells.

A novel DNA primase activity has been identified in HeLa cells infected with herpes simplex virus type 1 (HSV-1). Such an activity has not been detected in mock-infected cells. The primase activity coeluted with a portion of HSV-1 DNA polymerase from single-stranded DNA agarose columns loaded with high-salt extracts derived from infected cells. This DNA primase activity could be distinguished from host HeLa cell DNA primase by several criteria. First, the pH optimum of the HSV primase was relatively broad and peaked at 8.2 to 8.7 pH units. In contrast, the pH optimum of the HeLa DNA primase was very sharp and fell between pH 7.9 and 8.2. Second, freshly isolated HSV DNA primase was less salt sensitive than the HeLa primase and was eluted from single-stranded DNA agarose at higher salt concentrations than the host primase. Third, antibodies raised against individual peptides of the calf thymus DNA polymerase:primase complex cross-reacted with the HeLa primase but did not react with the HSV DNA primase. Fourth, freshly prepared HSV DNA primase appeared to be associated with the HSV polymerase, but after storage at 4 degrees C for several weeks, the DNA primase separated from the viral DNA polymerase. Separation or decoupling could also be achieved by gel filtration of the HSV polymerase:primase. This free DNA primase had an apparent molecular size of approximately 40 kilodaltons, whereas free HeLa DNA primase had an apparent molecular size of approximately 110 kilodaltons. On the basis of these data, we believe that the novel DNA primase activity in HSV-infected cells may be virus coded and that this enzyme represents a new and important function involved in the replication of HSV DNA.     

DNA polymerase-primase complex in wild-type and ts A1S9 mouse L-cells, temperature-sensitive for DNA replication during cell cycle progression

ts A1S9 mutant cells, derived from wild type WT-4 mouse L-cells, are temperature-sensitive (ts) for DNA synthesis and cell division. We try to determine the cause of the arrest of DNA replication in ts A1S9 cells at the nonpermissive temperature by comparing the modifications induced by the shift of temperature on the activity and the synthesis of DNA polymerase-alpha and DNA primase as a function of time. Forty-seven hours after temperature upshift DNA polymerase-alpha activity of ts A1S9 cells was inhibited by 90% while primase activity was barely detectable. By contrast, the activities of both enzymes increased to a plateau level in WT-4 cultured at either temperature and in ts A1S9 cells grown at the low permissive temperature. Study of the synthesis of DNA polymerase-alpha primase and of the structure of the enzyme complex during cell cycle progression was approached by immunoprecipitation of [35S]-labelled cells, with a specific monoclonal antibody directed against DNA polymerase-alpha. We have found that, irrespective of temperature of cultivation of WT-4 or ts A1S9 cells, this antibody precipitated polypeptides of 220, 186, 150, 110, 68-70, 60, and 48 kDa from cell extracts. With ts A1S9 cells cultivated at 38.5 degrees C for 48 hr the polypeptides of 220 and 186 kDa, associated with alpha-polymerase activity, were considerably more abundant than in the control cells, with a concomitant decline in the polypeptides of 60 and 48 kDa, implicated in primase activity. Thus the inhibition of DNA polymerase-alpha cannot be due to a decreased synthesis of the 186 kDa subunit but to its temperature inactivation. Consistent with a recent asymmetric dimeric model where polymerase-alpha complex and polymerase delta complex synthesize co-ordinately at the replication fork lagging and leading DNA strands, the observed alterations of polymerase-alpha and primase content explain the inhibition of DNA synthesis and the cell cycle arrest of the ts A1S9 cells at the nonpermissive temperature.