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.     

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.     

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.     

Characterization of the helicase and primase activities of the 63-kDa component of the bacteriophage T7 gene 4 protein

Leading and lagging strand DNA synthesis at the replication fork of bacteriophage T7 DNA requires the helicase and primase activities of the gene 4 protein. Gene 4 protein consists of two colinear polypeptides of 56- and 63-kDa molecular mass. We have demonstrated previously that the 56-kDa protein possesses helicase but lacks primase activity (Bernstein, J. A., and Richardson, C. C. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 396-400). The 63-kDa gene 4 protein has now been purified from extracts of T7-infected cells. The preparation contains 5-10% contaminating 56-kDa protein, as shown by Western analysis using polyclonal antibodies to the purified 56-kDa protein. The 63-kDa protein catalyzes DNA-dependent dTTP hydrolysis and has helicase activity; both specific activities are similar to those determined for the 56-kDa protein. The 63-kDa protein efficiently synthesizes sequence-specific di-, tri-, and tetraribonucleotides and stimulates the elongation of tetraribonucleotides by T7 DNA polymerase. Although the 56-kDa protein alone lacks primase activity, it enhances the primase activity of the 63-kDa protein 4-fold. This stimulation can be accounted for by a similar increase in the amount of primers synthesized by the 63-kDa protein in the presence of the 56-kDa protein.     

Purification and properties of a specific primase-stimulating factor of bovine thymus

The DNA replicase activity of the complex between bovine thymus DNA polymerase alpha and RNA primase was markedly decreased after the purification by ssDNA-cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose-step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat-labile and trypsin-sensitive, possessed a native molecular mass of approximately 150-200 kDa as judged by Sephacryl S-200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20-fold the replicase activity of purified DNA-polymerase-alpha-primase at a very low concentration (10 ng/50 microliter). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase-stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.     

The mouse DNA polymerase alpha-primase subunit p48 mediates species-specific replication of polyomavirus DNA in vitro.

Mouse cell extracts support vigorous replication of polyomavirus (Py) DNA in vitro, while human cell extracts do not. However, the addition of purified mouse DNA polymerase alpha-primase to human cell extracts renders them permissive for Py DNA replication, suggesting that mouse polymerase alpha-primase determines the species specificity of Py DNA replication. We set out to identify the subunit of mouse polymerase alpha-primase that mediates this species specificity. To this end, we cloned and expressed cDNAs encoding all four subunits of mouse and human polymerase alpha-primase. Purified recombinant mouse polymerase alpha-primase and a hybrid DNA polymerase alpha-primase complex composed of human subunits p180 and p68 and mouse subunits p58 and p48 supported Py DNA replication in human cell extracts depleted of polymerase alpha-primase, suggesting that the primase heterodimer or one of its subunits controls host specificity. To determine whether both mouse primase subunits were required, recombinant hybrid polymerase alpha-primases containing only one mouse primase subunit, p48 or p58, together with three human subunits, were assayed for Py replication activity. Only the hybrid containing mouse p48 efficiently replicated Py DNA in depleted human cell extracts. Moreover, in a purified initiation assay containing Py T antigen, replication protein A (RP-A) and topoisomerase I, only the hybrid polymerase alpha-primase containing the mouse p48 subunit initiated primer synthesis on Py origin DNA. Together, these results indicate that the p48 subunit is primarily responsible for the species specificity of Py DNA replication in vitro. Specific physical association of Py T antigen with purified recombinant DNA polymerase alpha-primase, mouse DNA primase heterodimer, and mouse p48 suggested that direct interactions between Py T antigen and primase could play a role in species-specific initiation of Py replication.     

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.     

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.     

Binding of the DNA polymerase alpha-DNA primase complex to the nuclear matrix in HeLa cells

It is well-known that there are multiple forms of DNA polymerase alpha. In order to determine which form(s) is (are) tightly bound, the activities were dissociated from DNA-poor nuclear matrices, with octyl beta-D-glucoside. Sucrose gradient sedimentation analysis revealed three bands with s values of 7.5, 10.5, and 13. The 7.5S form was free of DNA primase and represented only 10% of the total DNA polymerase alpha bound to the nuclear matrix. The 13S and the 10.5S forms each contained DNA primase activity. The 10.5S form comprised 85% of the DNA polymerase alpha activity and 95% of the DNA primase activity, dissociated from the nuclear matrix. Neither temperature of nuclease digestion nor various salt treatments of nuclei had significant effects on the proportions of DNA polymerase alpha and DNA primase activities bound to, or subsequently dissociated from, nuclear matrices. In a comparison of primase activity bound to the nuclear matrix, dissociated from the nuclear matrix, and in the soluble fraction, it was found that the bound activity had a lower ATP dependence, had less KCl inhibition, and was less sensitive to heat, compared to the dissociated and soluble activities. No differences in Mg2+ or pH dependence were noted. The amounts of DNA polymerase alpha and DNA primase activities bound to the nuclear matrix varied over the cell cycle of synchronized cells. Over the S phase, there were two peaks of matrix-bound DNA primase and two peaks of subsequently dissociated DNA polymerase alpha-DNA primase complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

A DNA primase from yeast. Purification and partial characterization

A DNA primase activity has been purified from the budding yeast Saccharomyces. The resulting preparation was nearly homogeneous and was devoid of DNA and RNA polymerase activities. The primase activity cofractionated with a Mr 65,000 polypeptide in sedimentation and chromatography procedures, and the native molecular weight of the enzyme corresponded closely to this value suggesting that the primase or an active proteolytic fragment of the protein exists as a monomer. Both heat-denatured calf thymus DNA and poly(dT) could be utilized by the enzyme as templates. Primase exhibited an absolute requirement for divalent cations and for rATP on a poly(dT) template. Although it required the ribonucleotide to initiate primer chains, the enzyme could incorporate the deoxynucleotide into primers. The product of the primase-catalyzed reaction was an oligonucleotide of discrete length (11-13 nucleotides), and oligonucleotides that were apparently dimers of this unit length were also observed. Primers that were synthesized were virtually identical in size in both the presence and absence of dATP incorporation. Although the bulk of DNA primase activity was isolated as a "free" enzyme, a portion of cellular primase activity co-chromatographed with DNA polymerase suggesting an association between these enzymes similar to that found in several higher eukaryotes.     

The mammalian primase is part of a high molecular weight DNA polymerase alpha polypeptide

The recently discovered eukaryotic primases have been found in tight association with certain DNA polymerase alpha forms. Here I present evidence that the high mol. wt. catalytic polypeptide (125,000) of an apparently homogeneous DNA polymerase alpha from freshly harvested calf thymus contains both polymerase and primase activity. This conclusion derives from the following three facts: (1) the two enzyme activities cannot be separated upon velocity sedimentation in 1.7 M urea, (2) both activities elute at a pI of 5.25 upon chromatofocussing and (3) after SDS-electrophoresis, renaturation of the enzymes in situ and measurement of DNA polymerase and primase activities in the gels, both enzymes have identical mobilities and coincide with the high mol. wt. catalytic subunit of DNA polymerase alpha.     

Activity levels of mouse DNA polymerase alpha-primase complex (DNA replicase) and DNA polymerase alpha, free from primase activity in synchronized cells, and a comparison of their catalytic properties

To asses the possible roles of the two active forms of mouse DNA polymerase alpha: primase--DNA-polymerase alpha complex (DNA replicase) and DNA polymerase alpha free from primase activity (7.3S polymerase), in nuclear DNA replication the correlation of their activity levels with the rate of nuclear DNA replication was determined and a comparison made of their catalytic properties. The experiments using either C3H2K cells, synchronized by serum starvation, or Ehrlich culture cells, arrested at the S phase by aphidicolin, showed DNA replicase to increase in cells in the S phase to at least six times that of the G0-phase cells but 7.3S polymerase to increase but slightly in this phase. This increase in DNA replicase activity most likely resulted from synthesis of a new enzyme, as shown by experiments using a specific monoclonal antibody, aphidicolin and cycloheximide. Not only with respect to the presence or absence of primase activity, but in other points as well the catalytic properties of these two forms were found to differ; DNA replicase preferred the activated calf thymus DNA with wide gaps of about 100 nucleotides long as a template-primer, while the optimal gap size for 7.3S polymerase was 40-50 nucleotides long. Size analysis of the products synthesized on M13 single-stranded circular DNA with a single 17-nucleotide primer by DNA replicase and 7.3S polymerase suggested the ability of DNA replicase to overcome a secondary structure formed in single-stranded DNA to be greater than that of 7.3S polymerase.     

The mechanism of action of an accessory protein for DNA polymerase alpha/primase

A previous paper reported the purification (from mouse cell extracts) and some of the properties of a protein, alpha accessory factor (AAF), that specifically stimulates DNA polymerase alpha/primase (1). We describe here studies on the mechanism of action of AAF. In the presence of AAF and a large excess of single-stranded circular DNA template, a molecule of DNA polymerase alpha/primase interacts with a single template DNA molecule priming and synthesizing multiple short DNA fragments covering thousands of nucleotides without detaching from the template, and, by many-fold repetition of the process, accomplishes serial replication of the population of DNA molecules. In contrast, without AAF the reaction involves the whole population of DNA molecules in parallel and with a very large number of binding events between DNA polymerase alpha/primase and DNA [corrected] template. The profound [corrected] increase in affinity of DNA polymerase alpha/primase for the DNA template that characterizes the mechanism suggests a functional identification of AAF as a template affinity protein. The resulting greater efficiency accounts for the ability of AAF to stimulate both the primase and polymerase activities of DNA polymerase alpha/primase. AAF also increases the processivity of DNA polymerase alpha/primase from approximately 15 to approximately 115 nucleotides, a size similar to that of mammalian Okazaki fragments, and it appears to allow DNA polymerase alpha/primase to traverse double-stranded regions of a DNA template. These features of the mechanism of AAF suggest that it may have a role in assisting DNA polymerase alpha/primase in synthesis of the lagging strand of a replication fork.     

Properties of the 3' to 5' exonuclease associated with porcine liver DNA polymerase gamma. Substrate specificity, product analysis, inhibition, and kinetics of terminal excision.

Porcine liver DNA polymerase gamma was shown previously to copurify with an associated 3' to 5' exonuclease activity (Kunkel, T. A., and Mosbaugh, D. W. (1989) Biochemistry 28, 988-995). The 3' to 5' exonuclease has now been characterized, and like the DNA polymerase activity, it has an absolute requirement for a divalent metal cation (Mg2+ or Mn2+), a relatively high NaCl and KCl optimum (150-200 mM), and an alkaline pH optimum between 7 and 10. The exonuclease has a 7.5-fold preference for single-stranded over double-stranded DNA, but it cannot excise 3'-terminal dideoxy-NMP residues from either substrate. Excision of 3'-terminally mismatched nucleotides was preferred approximately 5-fold over matched 3' termini, and the hydrolysis product from both was a deoxyribonucleoside 5'-monophosphate. The kinetics of 3'-terminal excision were measured at a single site on M13mp2 DNA for each of the 16 possible matched and mismatched primer.template combinations. As defined by the substrate specificity constant (Vmax/Km), each of the 12 mismatched substrates was preferred over the four matched substrates (A.T, T.A, C.G, G.C). Furthermore, the exonuclease could efficiently excise internally mismatched nucleotides up to 4 residues from the 3' end. DNA polymerase gamma was not found to possess detectable DNA primase, endonuclease, 5' to 3' exonuclease, RNase, or RNase H activities. The DNA polymerase and exonuclease activities exhibited dissimilar rates of heat inactivation and sensitivity to N-ethylmaleimide. After nondenaturing activity gel electrophoresis, the DNA polymerase and 3' to 5' exonuclease activities were partially resolved and detected in situ as separate species. A similar analysis on a denaturing activity gel identified catalytic polypeptides with molecular weights of 127,000, 60,000, and 32,000 which possessed only DNA polymerase gamma activity. Collectively, these results suggest that the polymerase and exonuclease activities reside in separate polypeptides, which could be derived from separate gene products or from proteolysis of a single gene product.     

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.     

Association of DNA primase with the beta/gamma subunits of DNA polymerase alpha from Drosophila melanogaster embryos

The DNA polymerase and primase activities of the intact DNA polymerase alpha from early embryos of Drosophila melanogaster co-sediment in native glycerol gradients. However, the activities are separated in glycerol gradients containing 2.8 M urea after treatment of the enzyme with 3.4 M urea. The 182,000-dalton alpha subunit which is required for DNA polymerase activity (Kaguni, L.S., Rossignol, J.-M., Conaway, R. C., and Lehman, I.R. (1983) Proc. Natl. Acad. Sci. U. S.A. 80, 2221-2225) is not required for DNA primase activity. Instead, primase activity resides in the 60,000-dalton (beta) and/or the 50,000-dalton (gamma) subunit. Neither polymerase nor primase has been found in association with the 73,000-dalton polypeptide which co-purifies with the intact enzyme.     

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     

Selective inhibition of DNA primase activity associated with DNA polymerase alpha from calf thymus

The photo-activatable analogs of ATP, 3'-O-(4-benzoyl) benzoic adenosine 5'-triphosphate (BzATP) and 8-azidoadenosine 5'-triphosphate (8-N3-ATP) were used to study the relationship between the polymerase activity and the closely associated primase activity of calf DNA polymerase alpha. A substantial loss of DNA primase activity occurred during pre-incubation and irradiation of DNA polymerase alpha with either BzATP or 8-N3-ATP. In contrast, polymerase activity was only slightly affected. In reactions carried out after pre-incubation with BzATP or 8-N3-ATP in the absence of UV illumination, inhibition was still observed, but it could be reversed by ATP. The specificity of the inhibition for primase activity, plus the ability of ATP to act as a antagonist of BzATP and 8-N3-ATP, suggest that effective interaction of these analogs with the multisubunit polymerase-primase complex is occurring uniquely at the active site of the DNA primase.