Mammalian cyclin/PCNA (DNA polymerase delta auxiliary protein) stimulates processive DNA synthesis by yeast DNA polymerase III.

Human cyclin/PCNA (proliferating cell nuclear antigen) is structurally, functionally, and immunologically homologous to the calf thymus auxiliary protein for DNA polymerase delta. This auxiliary protein has been investigated as a stimulatory factor for the nuclear DNA polymerases from S. cerevisiae. Calf cyclin/PCNA enhances by more than ten-fold the ability of DNA polymerase III to replicate templates with high template/primer ratios, e.g. poly(dA).oligo(dT) (40:1). The degree of stimulation increases with the template/primer ratio. At a high template/primer ratio, i.e. low primer density, cyclin/PCNA greatly increases processive DNA synthesis by DNA polymerase III. At low template/primer ratios (e.g. poly(dA).oligo(dT) (2.5:1), where addition of cyclin/PCNA only minimally increases the processivity of DNA polymerase III, a several-fold stimulation of total DNA synthesis is still observed. This indicates that cyclin/PCNA may also increase productive binding of DNA polymerase III to the template-primer and stabilize the template-primer-polymerase complex. The activity of yeast DNA polymerases I and II is not affected by addition of cyclin/PCNA. These results strengthen the hypothesis that yeast DNA polymerase III is functionally analogous to the mammalian DNA polymerase delta.     

Calf thymus DNA polymerase delta independent of proliferating cell nuclear antigen (PCNA).

DNA polymerase delta from calf thymus was purified under conditions that minimized proteolysis to a specific activity of 27,000 units/mg. The four step isolation procedure included phosphocellulose, hydroxyapatite, heparin-Sepharose and FPLC-MonoS. This enzyme consists of four polypeptides with Mr of 140, 125, 48 and 40 kilodaltons. Velocity gradient sedimentation in glycerol removed the 48 kDa polypeptide while the other three sedimented with the DNA polymerase activity. The biochemical properties of the three subunit enzyme and the copurification of 3'----5' exonuclease activity were typical for a bona fide DNA polymerase delta. Tryptic peptide analysis showed that the 140 kDa polypeptide was different from the catalytic 180 kDa polypeptide of calf thymus DNA polymerase alpha. Both high Mr polypeptides (140 and 125 kDa) were catalytically active as analysed in an activity gel. Four templates were used by DNA polymerase delta with different preferences, namely poly(dA)/oligo(dT)12-18 much much greater than activated DNA greater than poly(dA-dT) greater than primed single-stranded M13DNA. Calf thymus proliferating cell nuclear antigen (PCNA) could not stimulated this DNA polymerase delta in any step of the isolation procedure. If tested on poly(dA)/oligo(dT)12-18 (base ratio 10:1), PCNA had no stimulatory effect on DNA polymerase delta when tested with low enzyme DNA ratio nor did it change the kinetic behaviour of the enzyme. DNA polymerase delta itself did not contain PCNA. The enzyme had an intrinsic processivity of several thousand bases, when tested either on the homopolymer poly(dA)/oligo(dT)12-18 (base ratio 64:1) or on primed single-stranded M13DNA. Contrary to DNA polymerase alpha, no pausing sites were seen with DNA polymerase delta. Under optimal in vitro replication conditions the enzyme could convert primed single-stranded circular M13 DNA of 7,200 bases to its double-stranded form in less than 10 min. This supports that a PCNA independent DNA polymerase delta exists in calf thymus in addition to a PCNA dependent enzyme (Lee, M.Y.W.T. et al. (1984) Biochemistry 23, 1906-1913).     

Apparent stimulation of calf thymus DNA polymerase alpha by ATP.

The mechanism by which millimolar concentrations of ATP stimulate the activity and increase the processivity of calf thymus DNA polymerase alpha has been investigated with poly(dA)/oligo(dT) as template/primer to eliminate possible effects due to primer synthesis. The effect of ATP on the rate of DNA synthesis with this template/primer was found to be dependent upon whether or not the ATP was neutralized and the species of buffer used in the reaction. The present studies suggest that ATP stimulation of calf thymus DNA polymerase can be attributed to changes in the pH of the reaction mixture, a shift in the magnesium ion optimum, or both. Furthermore, effects of ATP on the processivity of DNA polymerase alpha could be mimicked by lowering the pH of the reaction mixture.     

Processiveness of DNA polymerases. A comparative study using a simple procedure.

In this communication, we describe a simple procedure for analyzing the processiveness of DNA polymerases in general. By choosing conditions for which the number of incorporations per available primer is less than 1, we have reduced the probability of a primer molecule being utilized by the enzyme more than once. The primer-template used was poly(dA)300:oligo(dT)10, and the product was isolated by oligo(dT)-cellulose chromatography. The number of dTMP residues added per association was determined from the [3H]dThd + [3'-3H]dTMP/[3H]dThd ratio of the product after its digestion by micrococcal nuclease and spleen phosphodiesterase. Using this procedure, we have found that Escherichia coli DNA polymerase I, T4 DNA polymerase, and calf thymus alpha- and beta-DNA polymerase are "quasi-processive." Most of these enzymes add on the average approximately 10 to 15 nucleotides before dissociating from the template. T5 DNA polymerase, on the other hand, is processive, i.e. it continues to replicate a given template until it is very close to the 5' end of the template. With "nicked DNA-like" poly(dA):oligo(dT), the processiveness of E. coli DNA polymerase I is increased 2- to 2.5-fold. The significance of this increase in determining the "patch size" during DNA repair is discussed.     

Polynucleotide recognition by DNA alpha-polymerase.

In a survey of template-primer preference of a mouse myeloma DNA alpha-polymerase, the fastest rate of DNA synthesis was with poly(dT) as template and (rA)24 as primer. Such a preference for poly(dT).oligo(rA) was not observed with other DNA polymerases of mouse origin. DNA synthesis in this system resulted in formation of oligo(dA) chains, not template-length poly(dA); thus, the average enzyme molecule bound to a poly(dT).(rA)24 complex and initiated a new oligo(dA) chain many times during the incubation. Binding experiments revealed that the alpha-polymerase had high affinity for poly(dT). Although the alpha-polymerase did not bind to poly(dl) and failed to replicate it inreactions with a base pair complementary primer, poly(dl) was replicated after a (dT) block had been grafted to its 3'-end and the oligo(rA) primer had been added. In similar experiments, the (dT) block was found to be much more effective than other 3'-terminal blocks in promoting replication of denatured calf thymus DNA. The results indicate that specific base sequences may regulate initiation of DNA syntehsis by this alpha-polymerase.     

The DNA sequence specificity of stimulation of DNA polymerases by factor D

The mechanism of enhancement of DNA polymerase activity by the murine DNA-binding protein factor D was investigated. Extension by Escherichia coli DNA polymerase I and calf thymus DNA polymerase-alpha of 5'-32P-labeled oligodeoxynucleotide primers that are complementary to poly(dT) or to bacteriophage M13 DNA was measured in the absence or presence of factor D. With 5'-[32P](dA)9.poly(dT), factor D enables E. coli polymerase I to fill approximately 15-nucleotide gaps between adjacent primers; whereas in the absence of the stimulatory protein, poly(dT) is not copied significantly. In order to study the nucleotide specificity of synthesis enhancement, we used M13mp10 DNA containing 4 consecutive thymidine residues downstream from the 3-hydroxyl terminus of an oligonucleotide primer. Upon addition of factor D, both polymerase I and polymerase-alpha can traverse this sequence more efficiently and thus generate longer DNA products. Densitometric analysis of nonextended and elongated 5'-32P-labeled M13 primer indicates that, without changing the frequency of primer utilization, factor D enhances the activity of these DNA polymerases by increasing their apparent processivity. By positioning oligonucleotide primers 4, 8, and 12 bases upstream from the (dT)4 template sequence, we show that the enhancement of synthesis by factor D is independent of the position of the oligothymidine cluster. We hypothesize that factor D interacts with oligo(dT).oligo(dA) domains in DNA to alter their conformation, which may normally obstruct the progression of DNA polymerases.     

Novel properties of DNA polymerase beta with poly(rA).oligo(dT) template-primer.

Purified DNA polymerase beta of calf thymus can utilize poly(rA).oligo(dT) as efficiently as poly(dA).oligo(dT) or activated DNA as a template primer. The poly(rA).oligo(dT)-dependent activity of DNA polymerase beta was found to differ markedly from the DNA-dependent activity of the same enzyme (with either activated calf thymus DNA or poly(dA).(dT)10) in the following respects. 1) Poly(rA)-dependent activity was strongly inhibited by natural DNA from various sources or synthetic deoxypolymer duplexes at very low concentrations (less than 0.5 microgram/ml) at which the DNA-dependent activity was affected to a much smaller extent, if at all. 2) Poly(rA)-dependent activity was inhibited by N-ethylmaleimide more strongly than DNA-dependent activity measured at 37 degrees C, while it was resistant to this reagent at 26 degrees C. 3) The curves of the activity versus substrate concentration were sigmoidal in the poly(rA)-dependent reaction but hyperbolic in the activated DNA-dependent reaction. A kinetic study suggested that the association of beta-enzyme protomers may be required to copy the poly(rA) strand.     

Calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis

We have demonstrated that calf thymus DNA polymerases alpha and delta are capable of highly processive DNA synthesis. Processivity values between 300 and 2000 nucleotides were observed when poly(dA)-oligo(dT) or singly primed single-stranded circular bacteriophage M13 DNA at pH 6.0 and 1 mM magnesium chloride was used. These conditions do not correlate with conditions, pH 7.0 and 5 mM magnesium chloride, that support the maximum synthetic rate. Lowering the pH and magnesium concentration lowers the Km value of the reaction with respect to primer terminus concentration. Furthermore, under these same conditions, both polymerases become insensitive to dissociation from the template as a result of encountering the 5' ends of primers. Overall, these results suggest that the affinity of the polymerases for the primer termini is higher throughout the polymerization reaction of pH and magnesium concentrations are lowered from those favoring maximum synthetic rate. Experiments with short primer templates, however, indicate that this higher affinity does not cause the DNA polymerase to remain stably bound after synthesizing up to the end of the template.     

A memory effect in DNA replication

A study of the polymerization/excision ratio in the replication of poly(dA), primed with oligo(dT), was carried out with E. coli DNA polymerase I, at various primer and enzyme concentrations. The variations in this ratio suggest that 1) the DNA polymerase is able to switch between two states of low and high exonuclease activities and 2) after dissociating from the template, the DNA polymerase drifts towards the low exonuclease state. The recovery of the high exonuclease state would require several successive incorporations.     

A mammalian DNA polymerase alpha holoenzyme functioning on defined in vivo-like templates.

In analogy to the Escherichia coli replicative DNA polymerase III we define two forms of DNA polymerase alpha: the core enzyme and the holoenzyme. The core enzyme is not able to elongate efficiently primed single-stranded DNA templates, in contrast to the holoenzyme which functions well on in vivo-like template. Using these criteria, we have identified and partially purified DNA polymerase alpha holoenzyme from calf thymus and have compared it to the corresponding homogeneous DNA polymerase alpha (defined as the core enzyme) from the same tissue. The holoenzyme is able to use single-stranded parvoviral DNA and M13 DNA with a single RNA primer as template. The core enzyme, on the other hand, although active on DNAs treated with deoxyribonuclease to create random gaps, is unable to act on these two long, single-stranded DNAs. E. coli DNA polymerase III holoenzyme also copies the two in vivo-like templates, while the core enzyme is virtually inactive. The homologous single-stranded DNA-binding proteins from calf thymus and from E. coli stimulate the respective holoenzymes and inhibit the core enzymes. These results suggest a cooperation between a DNA polymerase holoenzyme and its homologous single-stranded DNA-binding protein. The prokaryotic and the mammalian holoenzyme behave similarly in several chromatographic systems.     

Initiation of DNA synthesis by the calf thymus DNA polymerase-primase complex

The calf thymus DNA polymerase-alpha-primase complex purified by immunoaffinity chromatography catalyzes the synthesis of RNA initiators on phi X174 single-stranded viral DNA that are efficiently elongated by the DNA polymerase. Trace amounts of ATP and GTP are incorporated into products that are full length double-stranded circular DNAs. When synthetic polydeoxynucleotides are used as templates, initiation and DNA synthesis occurs with both poly(dT) and poly(dC), but neither initiation nor DNA synthesis was observed with poly(dA) and poly(dI) templates. Nitrocellulose filter binding and sucrose gradient centrifugation studies show that the DNA polymerase-primase complex binds to deoxypyrimidine polymers, but not to deoxypurine polymers. Using d(pA)-50 with 3'-oligo(dC) tails and d(pI)-50 with 3'-oligo(dT) tails, initiator synthesis and incorporation of deoxynucleotide can be demonstrated when the average pyrimidine sequence lengths are 8 and 4, respectively. These results suggest that purine polydeoxynucleotides are used as templates by the DNA polymerase only after initiation has occurred on the oligodeoxypyrimidine sequence and that the pyrimidine stretch required by the primase activity is relatively short. Analysis of initiator chain length with poly(dC) as template showed a series of oligo(G) initiators of 19-27 nucleotides in the absence of dGTP, and 5-13 nucleotides in the presence of dGTP. The chain length of initiators synthesized by the complex when poly(dT) or oligodeoxythymidylate-tailed poly(dI) was used can be as short as a dinucleotide. Analysis of the products of replication of oligo(dC)-tailed poly(dA) shows that initiator with chain length as low as 4 can be used for initiation by the polymerase-primase complex.     

Studies on the processivity of highly purified calf thymus 8S and 7.3S DNA polymerase alpha

Template-challenge experiments indicate no gross difference in processivity of the calf thymus DNA polymerase α A and C enzymes. Both enzymes appear to be distributive. Results showing the apparent processive nature of both enzymes on poly (dC). oligo (dG)₁₀ when challenged with poly (dA). oligo (dT)₁₀ are explicable by the failure of both enzymes to bind to the challenging template rather than by the presence of an initiation factor which preferentially binds to certain templates.     

Overexpression, purification, and characterization of Escherichia coli bacteriophage PRD1 DNA polymerase. In vitro synthesis of full-length PRD1 DNA with purified proteins.

The bacteriophage PRD1 DNA polymerase gene (gene I) has been cloned into the expression vector pPLH101 under the control of the lambda pL promoter. Tailoring of an efficient ribosome binding site in front of the gene by polymerase chain reaction led to a high level heat-inducible expression of the corresponding gene product (P1) in Escherichia coli cells. Expression was confirmed in vivo by complementation of phage PRD1 DNA polymerase gene mutants and in vitro by formation of the genome terminal protein P8-dGMP replication initiation complex. Expressed PRD1 DNA polymerase was purified to apparent homogeneity in an active form. DNA polymerase, 3'-5'-exonuclease, and P8-dGMP replication initiation complex formation activities cosedimented in glycerol gradient with a protein of 65 kDa, the size expected for PRD1 DNA polymerase. The DNA polymerase was active on DNase I-activated calf thymus DNA, poly(dA).oligo(dT) and poly(dA-dT) primer/templates as well as on native phage PRD1 genome. The 3'-5'-exonuclease activity was specific for single-stranded DNA and released mononucleotides. No 5'-3'-exonuclease activity was detected. The inhibitor/activator spectrum of the PRD1 DNA polymerase was also studied. An in vitro replication system with purified components for bacteriophage PRD1 was established. Formation of the P8-dGMP replication initiation complex was a prerequisite for phage DNA replication, which proceeded from the initiation complex and yielded genome length replication products.     

Autographa californica nuclear polyhedrosis virus DNA polymerase: measurements of processivity and strand displacement

The DNA polymerase (DNApol) of Autographa californica nuclear polyhedrosis virus was purified to homogeneity from recombinant baculovirus-infected cells. DNApol was active in polymerase assays on singly primed M13 template, and full-length replicative form II product was synthesized at equimolar ratios of enzyme to template. The purified recombinant DNApol was shown to be processive by template challenge assay. Furthermore, DNApol was able to incorporate hundreds of nucleotides on an oligo(dT)-primed poly(dA) template with limiting amounts of polymerase. DNApol has moderate strand displacement activity, as it was active on nicked and gapped templates, and displaced a primer in a replication-dependent manner. Addition of saturating amounts of LEF-3, the viral single-stranded DNA-binding protein (SSB), increased the innate strand displacement ability of DNApol. However, when LEF-3 was added prior to the polymerase, it failed to stimulate DNApol replication on a singly primed M13 template because the helix-destabilizing activity of LEF-3 caused the primer to dissociate from the template. Escherichia coli SSB efficiently substituted for LEF-3 in the replication of a nicked template, suggesting that specific protein-protein interactions were not required for strand displacement in this assay.     

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.     

Biochemical and functional comparison of DNA polymerases alpha, delta, and epsilon from calf thymus.

DNA polymerase alpha, delta and epsilon can be isolated simultaneously from calf thymus. DNA polymerase delta was purified to apparent homogeneity by a four-column procedure including DEAE-Sephacel, phenyl-Sepharose, phosphocellulose, and hydroxylapatite, yielding two polypeptides of 125 and 48 kDa, respectively. On hydroxylapatite DNA polymerase delta can completely be separated from DNA polymerase epsilon. By KCl DNA polymerase delta is eluted first, while addition of potassium phosphate elutes DNA polymerase epsilon. DNA polymerases delta and epsilon could be distinguished from DNA polymerase alpha by their (i) resistance to the monoclonal antibody SJK 132-20, (ii) relative resistance to N2-[p-(n-butyl)phenyl]-2-deoxyguanosine triphosphate and 2-[p-(n-butyl)anilino]-2-deoxyadenosine triphosphate, (iii) presence of a 3'----5' exonuclease, (iv) polypeptide composition, (v) template requirements, (vi) processivities on the homopolymer poly(dA)/oligo(dT12-18), and (vii) lack of primase. The following differences of DNA polymerase delta to DNA polymerase epsilon were evident: (i) the independence of DNA polymerase epsilon to proliferating cell nuclear antigen for processivity, (ii) utilization of deoxy- and ribonucleotide primers, (iii) template requirements in the absence of proliferating cell nuclear antigen, (iv) mode of elution from hydroxylapatite, and (v) sensitivity to d2TTP and to dimethyl sulfoxide. Both enzymes contain a 3'----5' exonuclease, but are devoid of endonuclease, RNase H, DNA helicase, DNA dependent ATPase, DNA primase, and poly(ADP-ribose) polymerase. DNA polymerase delta is 100-150 fold dependent on proliferating cell nuclear antigen for activity and processivity on poly(dA)/oligo(dT12-18) at base ratios between 1:1 to 100:1. The activity of DNA polymerase delta requires an acidic pH of 6.5 and is also found on poly(dT)/oligo(dA12-18) and on poly(dT)/oligo(A12-18) but not on 10 other templates tested. All three DNA polymerases can be classified according to the revised nomenclature for eukaryotic DNA polymerases (Burgers, P.M. J., Bambara, R. A., Campbell, J. L., Chang, L. M. S., Downey, K. M., Hübscher, U., Lee, M. Y. W. T., Linn, S. M., So, A. G., and Spadari, S. (1990) Eur. J. Biochem. 191, 617-618).     

Mammalian DNA polymerase alpha: a replication competent holoenzyme form from calf thymus.

A complex "replication competent" holoenzyme form of DNA polymerase alpha (RC-alpha) was purified 10,000 fold from calf thymus through the use of an assay employing primed single stranded circular DNA template. The RC-alpha form could partially replicate a double-stranded oligo(dT)-tailed linear DNA and could completely convert primed single-stranded circular DNA to its double stranded form. The RC-alpha was resolved by denaturing gel electrophoresis into at least 10 discrete polypeptide species ranging in apparent molecular mass from 200 to 47 kilodaltons; three of the bands (apparent Mr of 200, 118 and 63 kilodaltons) displayed DNA polymerase activity in denaturing gel activity assay. The isolation of RC-alpha required the use of absolutely fresh calf thymus, the inclusion of ATP and protease inhibitors throughout the purification procedure. Treatment of the RC-alpha with the neutralizing anti-DNA polymerase alpha monoclonal antibody SJK 132-20 (Tanaka et al. (1982), J. Biol. Chem. 257, 8386-8390) in nondenaturing conditions selected the complete set of 10 polypeptides, whereas treatment in denaturing conditions selected the 200 kilodalton catalytic DNA polymerase active polypeptide. The properties and the behaviour of the RC-alpha preparation following removal of specific polypeptides strongly suggested that the capacity of RC-alpha to extend and replicate long template requires the function of nonproteolysed form of the 200 kilodaltons catalytic DNA polymerase core and at least 6 other auxiliary polypeptides of, respectively, 98, 87, 63, 54, 49 and 47 kilodaltons.     

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.