Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties

Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.     

HeLa DNA polymerase alpha activity in vitro: specific stimulation by a non-enzymic protein factor.

A non-enzymic protein factor that increases the in vitro rate of synthesis by HeLa DNA polymerase alpha 15- to 30-fold with denatured DNA as template has been partially purified from the cytoplasmic fraction of HeLa cells. The stimulatory effect is highly specific for HeLa DNA polymerase alpha and for DNA templates that contain extensive regions of single-strandedness. Synthesis with denatured DNA as template presumably proceeds from 3'-hydroxyl termini formed at loop-back regions since the synthesized DNA product and template are covalently linked. The stimulatory protein factor chromatographs as a basic protein, has an approximate molecular weight of 30,000 daltons and binds with moderate affinity to denatured DNA cellulose, being eluted by o.4M NaCl. The purified factor lacks detectable DNA polymerase, exo- and endodeoxyribonuclease and RNA polymerase activities. It also does not promote helix-coil transitions with poly[d(A-T)] and Clostridium perfringens DNA.     

Template-selective stimulation of diverse DNA polymerases by the murine DNA-binding protein factor D

Factor D, a template-selective DNA polymerase-alpha stimulatory protein from mouse liver (Fry, M., Lapidot, J., and Weisman-Shomer, P. (1985) Biochemistry 24, 7549-7556) is shown here to enhance the activities of diverse DNA polymerases with a cognate template specificity. DNA synthesis catalyzed by Escherichia coli DNA polymerase I, avian myeloblastosis virus polymerase, and some mammalian alpha- and gamma-polymerases was increased by factor D. With every enhanced polymerase, factor D increased the rate of copying of only poly(dT) among various tested synthetic poly-deoxynucleotides. Of the natural DNA templates examined, rates of copying of sparsely primed denatured DNA and of singly primed circular phi X174 or M13 bacteriophage DNA, but not of activated DNA, were enhanced. Michaelis constants (Km) of affected templates with responsive polymerases were decreased by factor D, without alteration in maximum velocity (Vmax). By contrast, factor D increased Vmax of deoxyribonucleoside 5'-monophosphate incorporation without changing Km of deoxyribonucleoside 5'-triphosphate substrates. Binding of factor D to poly(dT), poly(dA).poly(dT), and DNA, but less to poly(dA), was indicated by specific retention of their complexes on a DEAE-cellulose column. That factor D does not bind to DNA polymerase-alpha or to its complex with the DNA template was demonstrated by the failure of the factor to be coprecipitated with alpha-polymerase by anti-polymerase-alpha monoclonal antibodies in either the absence or presence of various templates. Lack of binding of factor D to the polymerase molecule was also indicated by simultaneous maximum stimulation of two competing polymerases by a limiting amount of factor. These combined results suggest that the enhancement of DNA synthesis is exerted through interaction of factor D with the template. It is proposed that this association leads to a tighter binding of the polymerase to the template and facilitates DNA synthesis.     

Rabbit liver factor D, a poly(thymidine) template stimulatory protein of DNA polymerases: purification and characterization

Factor D, a DNA binding protein that enhances the activities of diverse DNA polymerases with a common restricted set of templates, was initially characterized in mouse liver but has resisted extensive purification. In this paper, we report that a similar stimulatory activity can be obtained in highly purified form from nuclei of rabbit hepatocytes. The rabbit liver protein increases the rates at which several DNA polymerases copy sparsely primed natural DNA templates and primed synthetic poly(dT), but it has no effect on the rates of copying of activated DNA or of poly(dG), poly(dA), and poly(dC). Direct binding of the purified stimulatory protein to an oligomer that contains a (dT)16 base stretch is visualized by retardation of the nucleoprotein complex on nondenaturing electrophoretograms. In the presence of the enhancing factor, Michaelis constants, Km, of responsive polymerase for singly primed bacteriophage M13 DNA and for poly(dT), but not for poly(dA), are decreased. Product analysis of M13 DNA primer extension indicates that the rabbit factor augments the apparent processivity of DNA polymerase by decreasing the extent of enzyme pausing at a tract of four consecutive thymidine residues in the template. Gel filtration of the native stimulatory protein yields an apparent relative molecular size of 58 +/- 2 kilodaltons. Stimulatory activity is readily inactivated by heat or by trypsin digestion, but it is resistant to micrococcal nuclease, N-ethyl-maleimide, or calcium ions.     

Factor C from rabbit liver. A new poly(dC) and poly[d(G-C)] template-selective stimulatory protein of DNA polymerases

We have undertaken a search for mammalian DNA-binding proteins that enhance the activity of DNA polymerases in a template sequence-specific fashion. In this paper, we report the extensive purification and characterization of a new DNA-binding protein from rabbit liver that selectively stimulates DNA polymerases to copy synthetic poly[d(G-C)] and the poly(dC) strand of poly(dC).poly(dG) as well as single-stranded natural DNA that contains stretches of oligo(dC). The enhancing protein, a polypeptide of 65 kDa designated factor C, stimulates the copying of the two synthetic templates by Escherichia coli DNA polymerase I, Micrococcus luteus polymerase, and eukaryotic DNA polymerases alpha and beta, but not by avian myeloblastosis virus polymerase. Factor C, however, does not affect utilization by these polymerases of the poly(dG) strand of poly(dC).poly(dG), of poly(dC) primed by oligo(dG), or of poly(dA).poly(dT) and poly[d(A-T)]. With polymerase I, Michaelis constants (Km) of poly[d(G-C)] and of the poly(dC) strand of poly(dC).poly(dG) are decreased by factor C 37- and 4.7-fold, respectively, whereas maximum velocity (Vmax) remains unchanged. By contrast, neither the Km value of the poly(dG) strand of poly(dC).poly(dG) nor the Vmax value with this template is altered by factor C. Rates of copying of activated DNA, denatured DNA, or singly primed M13 DNA are not affected significantly by factor C. However, primer extension analysis of the copying of recombinant M13N4 DNA that contains runs of oligo(dC) within an inserted thymidine kinase gene shows that factor C increases processivity by specifically augmenting the efficiency at which polymerase I traverses the oligo(dC) stretches. Direct binding of factor C to denatured DNA is indicated by retention of the protein-DNA complex on columns of DEAE-cellulose. Binding of factor C to poly[d(G-C)] is demonstrated by the specific adsorption of the enhancing protein to columns of poly[d(G-C)]-Sepharose. We propose that by binding to poly[d(G-C)] and to poly(dC).poly(dG), factor C enables tighter binding of some DNA polymerases to these templates and facilitates enzymatic activity.     

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 auxiliary protein for DNA polymerase-delta from fetal calf thymus

An auxiliary protein which affects the ability of calf thymus DNA polymerase-delta to utilize template/primers containing long stretches of single-stranded template has been purified to homogeneity from the same tissue. The auxiliary protein coelutes with DNA polymerase-delta on DEAE-cellulose and phenyl-agarose chromatography but is separated from the polymerase on phosphocellulose chromatography. The physical and functional properties of the auxiliary protein strongly resemble those of the beta subunit of Escherichia coli DNA polymerase III holoenzyme. A molecular weight of 75,000 has been calculated from a sedimentation coefficient of 5.0 s and a Stokes radius of 36.5 A. A single band of 37,000 daltons is seen on sodium dodecyl sulfate gel electrophoresis, suggesting that the protein exists as a dimer of identical subunits. The purified protein has no detectable DNA polymerase, primase, ATPase, or nuclease activity. The ability of DNA polymerase-delta to replicate gapped duplex DNA is relatively unaffected by the presence of the auxiliary protein, however, it is required to replicate templates with low primer/template ratios, e.g. poly(dA)/oligo(dT) (20:1), primed M13 DNA, and denatured calf thymus DNA. The auxiliary protein is specific for DNA polymerase-delta; it has no effect on the activity of calf thymus DNA polymerase-alpha or the Klenow fragment of E. coli DNA polymerase I with primed homopolymer templates. Although the auxiliary protein does not bind to either single-stranded or double-stranded DNA, it does increase the binding of DNA polymerase-delta to poly(dA)/oligo(dT), suggesting that the auxiliary protein interacts with the polymerase in the presence of template/primer, stabilizing the polymerase-template/primer complex.     

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.     

A complex between replication factor A (SSB) and DNA helicase stimulates DNA synthesis of DNA polymerase alpha on double-stranded DNA.

A helicase-like DNA unwinding activity was found in highly purified fractions of the calf thymus single-stranded DNA binding protein (ctSSB), also known as replication protein A (RP-A) or replication factor A (RF-A). This activity depended on the hydrolysis of ATP or dATP, and used CTP with a lower efficiency. ctSSB promoted the homologous DNA polymerase alpha to perform DNA synthesis on double-stranded templates containing replication fork-like structures. The rate and amount of DNA synthesis was found to be dependent on the concentration of ctSSB. At a 10-fold mass excess of ctSSB over double-stranded DNA, products of 200-600 nucleotides in length were obtained. This comprises or even exceeds the length of a eukaryotic Okazaki fragment. The ctSSB-associated DNA helicase activity is most likely a distinct protein rather than an inherent property of SSB, as inferred from titration experiments between SSB and DNA. The association of a helicase with SSB and the stimulatory action of this complex to the DNA polymerase alpha-catalyzed synthesis of double-stranded DNA suggests a cooperative function of the three enzymatic activities in the process of eukaryotic DNA replication.     

Template specific inhibitor of mammalian DNA polymerases.

A study of the inhibition of mouse cellular DNA polymerases by poly-nucleotides and their vinyl analogs is presented. Poly(dT)-directed poly(dA) synthesis by representatives of all three classes of cellular DNA polymerase could be completely inhibited by poly(9-vinyladenine), although higher concentrations were required in the case of the gamma class enzyme. Studies on the mechanism of the inhibition using the alpha class DNA polymerase and different templates showed that the enzyme activity was inhibited in all cases where base-pairing between the vinyl polymer and the template occurred; poly(9-vinyladenine) did not interfere with the replication of templates to which it does not bind. The inhibition occurred shortly after addition of poly(9-vinyladenine) to ongoing reactions, yet the enzyme was not displaced from the template - primer complex.     

Highly efficient copying of single-stranded DNA by eukaryotic cell chromatin.

Chromatin prepared from S phase hepatoma tissue culture (HTC) cell incorporates in vitro about 11-14 pmoles [3H]dTMP into DNA in 30 min. Single-stranded DNA added to this chromatin stimulates DNA synthesis more than 40-fold whereas activated DNA enhances it about 60-fold. By contrast, stimulation of DNA synthesis by activated DNA in a crude nuclear extract exceeds the stimulation exerted by denatured DNA by a factor of 7. Stimulation of DNA synthesis by denatured DNA is not due to stabilization of either the chromatin or the product of the endogenous reaction. On the other hand, we find that poly(dC) and poly (dT) enhance DNA synthesis by serving as templates which are copied by chromatin in a true complementary fashion. It seems therefore, that eukaryotic cell chromatin is able to copy single-stranded DNA at a high efficiency. Chromatin of G1 arrested cell copies exogenous templates at a considerably reduced rate. The enzyme responsible for the copying of denatured DNA is tentatively identified as DNA polymerase alpha on the basis of its sensitivity to sulfhydril group blocking, its requirements for ions and failure to copy the ribo strand of oligo(dT) poly(A).     

Detection and characterization of a novel factor that stimulates DNA polymerase alpha

A novel factor that stimulates DNA polymerase alpha activity on poly(dA) X oligo(dT) has been identified and partially purified from mouse FM3A cells. The assay system for the factor contained poly(ethylene glycol) 6000. The activities of DNA polymerase alpha on poly(dA) X oligo(dT) in the presence and absence of the stimulating factor were increased greatly by the addition of poly(ethylene glycol). Stimulation by the factor was observed at all the primer to template ratios tested from 0.01 to 0.3. The highest activity was observed at the ratio of 0.05, corresponding to about 3.3 primers on one template in the presence of the factor. The concentration of DNA polymerase alpha used in the assay affected the stimulation by the factor, and the stimulation became more prominent at concentrations of the enzyme lower than 0.04 unit per assay. The stimulating factor lowered the Km value of DNA polymerase alpha for the template-primer, though they had no effect on the Km value for dTTP substrate. The results of product analysis suggested that the stimulation by the factor is mainly due to the increase in the initiation frequency of DNA synthesis from the primers. The stimulating factor specifically stimulated DNA polymerase alpha but not DNA polymerases beta and gamma. Furthermore, the factor formed a complex with DNA polymerase alpha under a certain condition.     

Infidelity of DNA synthesis by reverse transcriptase

The fidelity of purified DNA polymerase from avian myeloblastosis virus in precisely copying polynucleotide templates was determined. With poly (dA-dT) · poly (dA-dT) as a template, one molecule of the incorrect basepaired nucleotide (dCTP) is incorporated for every 6000 nucleotides polymerized. When copying the ribo strand of poly (rA) · poly (dT) the error rate is approximately one in 600. It is suggested that the enzyme makes similar errors $\text{in}\text{vivo}$ and thus could be mutagenic.     

DNA polymerase alpha cofactors C1C2 function as primer recognition proteins

Most, if not all, of the DNA polymerase alpha activity in monkey and human cells was complexed with at least two proteins, C1 and C2, that together stimulated the activity of this enzyme from 180- to 1800-fold on low concentrations of denatured DNA, parvovirus DNA, M13, and phi X174 DNA or RNA-primed DNA templates, and poly(dT):oligo(dA) or oligo(rA). These primer-template combinations, which have from 200 to 5000 bases of template/primer, were then 7- to 50-fold more effective as substrates than DNase I-activated DNA. C1C2 specifically stimulated alpha polymerase, and only from the same cell type. Alpha X C1C2-polymerase reconstituted from purified alpha polymerase and the C1C2 cofactor complex behaved the same as native alpha X C1C2-polymerase and C1C2 had no effect on the sensitivity of alpha polymerase to aphidicolin, dideoxythymidine triphosphate, and N-ethylmaleimide. In the presence of substrates with a high ratio of single-stranded DNA template to either DNA or RNA primar, C1C2 increased the rate of DNA synthesis by decreasing the Km for the DNA substrate, decreasing the Km for the primer itself, increasing the use of shorter primers, and stimulating incorporation of the first deoxyribonucleotide. In contrast, C1C2 had no effect on the Km values for deoxyribonucleotide substrates (which were about 150-fold higher than for DNA replication in isolated nuclei), the ability of specific DNA sequences to arrest alpha polymerase, or the processivity of alpha polymerase. Accordingly, C1C2 function as primer recognition proteins. However, C1C2 did not reduce the comparatively high Km values or stimulate DNA synthesis by alpha polymerase on lambda DNA ends and DNase I-activated DNA, substrates with 12 and about 30-70 bases of template/primer, respectively. DNA restriction fragments with 1 to 4 bases of template/primer were substrates for neither alpha nor alpha X C1C2-polymerase. Therefore, we propose that C1C2 enhances the ability of alpha polymerase to initiate DNA synthesis by eliminating nonproductive binding of the enzyme to single-stranded DNA, allowing it to slide along the template until it recognizes a primer.     

Characterization of an alpha-like DNA polymerase from Bombyx mori silkglands.

A soluble DNA polymerase has been purified near to homogeneity from Bombyx mori silkglands. The following characteristics were observed: high molecular weight (about 150 000 - 220 00); optimum pH about 8; inhibition by high salt concentrations, sulfhydryl-group blocking agents and polyamines; absence of nuclease activity; preference for magnesium as required divalent cation with all the efficient template-primers tested; and clear template-primer specificity, the purified enzyme being able to copy primed - polydeoxyribonucleotide templates [activated DNA, poly(dA).oligo(dT), poly(dA).oligo(rU)] but not polyribonucleotide chains [poly(rA).oligo(dT), poly(rA).oligo(rU)] in the presence of either Mg++ or MN++. Believed to represent the bulk of silkgland DNA polymerase activity, the purified soluble enzyme most resembles vertebrate DNA polymerases alpha when it is compared to other eukaryotic DNA polymerases as yet characterized.     

Isolation of the DNA polymerase alpha core enzyme from mouse cells

DNA polymerase alpha has been purified from mouse hybridoma cells approximately 30,000-fold using a combination of conventional and high performance liquid chromatography. In contrast to previous characterizations of mammalian DNA polymerase alpha, this enzyme has a single high molecular mass polypeptide (185 kDa) in tight association with a 68-kDa polypeptide and this structure appears to be the core DNA polymerase of the mouse cells. The biochemically purified enzyme, with a specific activity of approximately 200,000 units/mg protein, has an estimated molecular mass by gel filtration chromatography of 240 kDa and sedimentation value of 9 S, consistent with the enzyme being a heterodimer of 185 and 68 kDa. The enzyme is sensitive to both N-ethylmaleimide and aphidicolin and insensitive to ddTTP. Using an activated DNA template, the apparent Km values for the deoxynucleotide triphosphates are approximately 0.5-1 microM. The purified DNA polymerase has neither exonuclease nor primase activities and is the predominant DNA polymerase alpha activity in the mouse cells.     

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.     

Purification and characterization of two forms of DNA polymerase alpha from mouse FM3A cells: a DNA polymerase alpha-primase complex and a free DNA polymerase alpha

Two forms of DNA polymerase alpha, alpha 1 and alpha 2, have been partially purified from mouse FM3A cells by discriminating one form from the other on the basis of the association of primase activity. The primase activity in the most purified alpha 1 fraction co-sedimented with the DNA polymerase activity in a glycerol gradient, and almost no primase activity was detected in the most purified alpha 2 fraction. The primase activity associated with DNA polymerase alpha was assayed indirectly by measuring ATP-dependent DNA synthesis with poly (dT) as template. Characterization of the assay system was performed with the purified alpha 1. The system was absolutely dependent on the presence of ATP and a divalent cation. Mn2+ was much more effective than Mg2+, and 5-fold higher activity was observed with Mn2+ than with Mg2+ at their optimal concentrations. The primase activity assayed by the above system showed sensitivity to (NH4)2SO4 very similar to that of free primase reported by Tseng and Ahlem (J. Biol. Chem. 258, 9845-9849, 1983). The activity was inhibited by more than 50% by 20 mM (NH4)2SO4. alpha 1 and alpha 2 were very similar as DNA polymerases in their sensitivity to several inhibitors and their preference for template-primers, except that alpha 1 had a slightly greater preference for poly (dT) X (rA)10 than alpha 2 did. The major difference between the two forms was observed in their S values, 8.2 and 6.4 S for alpha 1 and alpha 2, respectively.     

On the DNA polymerase III of mouse myeloma: partial purification and characterization.

A high molecular weight membrane-bound DNA polymerase from the mouse myeloma, MOPC-104E, has been purified extensively, and characterized with regard to physical and reaction properties. This enzyme, which is readily distinguishable from other myeloma enzymes that are analogous to the recognized forms of cellular DNA polymerase, is ddesignated DNA polymerase III. DNA polymerase III activity in whole homogenates from MOPC-104E was solubilized and then prurifed using a series of ion-exchange chromatographic procedures followed by DNA-cellulose chromatography and glycerol gradient centrifugation; the enzyme activity as measured with poly(rA)-(dT)12-18 as template-primer and Mn2+ as divalent cation, was purified as much as 18,000-fold. In the final stages of the pruification, DNA polymerase III possessed no detectable RNA polymerase activity, nucleoside diphosphokinase activity, or nucease activity toward DNA or single- and double-stranded RNA...