Model RNA-directed DNA synthesis by avian myeloblastosis virus DNA polymerase and its associated RNase H.

A model RNA template-primer system is described for the study of RNA-directed double-stranded DNA synthesis by purified avian myeloblastosis virus DNA polymerase and its associated RNase H. In the presence of complementary RNA primer, oligo(rI), and the deoxyribonucleoside triphosphates dGTP, dTTP, and dATP, 3'-(rC)30-40-poly(rA) directs the sequential synthesis of poly(dT) and poly(dA) from a specific site at the 3' end of the RNA template. With this model RNA template-primer, optimal conditions for double-stranded DNA synthesis are described. Analysis of the kinetics of DNA synthesis shows that initially there is rapid synthesis of poly(dT). After a brief time lag, poly(dA) synthesis and the DNA polymerase-associated RNase H activity are initiated. While poly(rA) is directing the synthesis of poly(dT), the requirements for DNA synthesis indicate that the newly synthesized poly(dT) is acting as template for poly(dA) synthesis. Furthermore, selective inhibitor studies using NaF show that activation of RNase H is not just a time-related event, but is required for synthesis of the anti-complementary strand of DNA. To determine the specific role of RNase H in this synthetic sequence, the primer for poly(dA) synthesis was investigated. By use of formamide--poly-acrylamide slab gel electrophoresis, it is shown that poly(dT) is not acting as both template and primer for poly(dA) synthesis since no poly(dT)-poly(dA) covalent linkages are observed in radioactive poly(dA) product. Identification of 2',3'-[32P]AMP on paper chromatograms of alkali-treated poly(dA) product synthesized with [alpha-32P]dATP as substrate demonstrates the presence of rAMP-dAMP phosphodiester linkages in the poly(dA) product. Therefore, a new functional role of RNase H is demonstrated in the RNA-directed synthesis of double-stranded DNA. Not only is RNase H responsible for the degradation of poly(rA) following formation of a poly(rA)-poly(dT) hybrid but also the poly(rA)fragments generated are serving as primers for initiation of synthesis of the second strand of the double-stranded DNA.     

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.     

Effect of ribonuclease H from chick embryo on the covalent-linked poly(A)--poly(dA) complementary to poly(dT) template

In vitro poly(dA) synthesis on poly(dT) template can be initiated by poly(A) primer. Poly(A) chains are covalently extended by DNA polymerase. The reaction product consists of poly(dA) chain with poly(A) at their 5'-ends, hydrogen bonded to the template poly(dT). The primer poly(A) is linked to the product poly(dA) via a 3':5'-phosphodiester bond, and can be specifically removed by ribonuclease H from chick embryos, leaving a 5'-phosphate end of poly(dA). Poly- or oligoriboadenylate longer than the (pA)5 could serve as a priming activity to synthesize poly(A) covalently linked to poly(dA).     

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.     

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.     

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.     

Distamycin paradoxically stimulates the copying of oligo(dA).poly(dT) by DNA polymerases

Distamycin A, a polypeptide antibiotic, binds to dA.dT-rich regions in the minor groove of B-DNA. By virtue of its nonintercalating binding, distamycin acts as a potent inhibitor of the synthesis of DNA both in vivo and in vitro. Here we report that distamycin paradoxically stimulates Escherichia coli DNA polymerase I (pol I), its large (Klenow) fragment, and bacteriophage T4 DNA polymerase to copy oligo(dA).poly(dT) in vitro. It is found that distamycin increases the maximum velocity (Vmax) of the extension of the oligo(dA) primer by pol I without affecting the Michaelis constant (Km) of the primer. Gel electrophoresis of the extended primer indicates that the antibiotic specifically increases the rate of addition of the first three dAMP residues. Lastly, in the presence of both distamycin and the oligo(dT)-binding protein factor D, which increases the processivity of pol I, a synergistic stimulation of polymerization is attained. Taken together, these results suggest that distamycin stimulates synthesis by increasing the rate of initiation of oligo(dA) extension. The stimulatory effect of distamycin is inversely related to the stability of the primer-template complex. Thus, maximum stimulation is exerted at elevated temperatures and with shorter oligo(dA) primers. That distamycin increases the thermal stability of [32P](dA)9.poly(dT) is directly demonstrated by electrophoretic separation of the hybrid from dissociated [32P](dA)9 primer. It is proposed that by binding to the short primer-template duplex, distamycin stabilizes the oligo(dA).poly(dT) complex and, therefore, increases the rate of productive initiations of synthesis at the primer terminus.     

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.     

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.     

Studies on the inhibition of highly purified calf thymus 8S and 7.3S DNA polymerase alpha by aphidicolin.

On activated DNA aphidicolin competitively inhibits the incorporation of dCMP by both calf thymus DNA polymerase alpha A2 and C enzymes and inhibits the incorporation of the other three deoxynucleoside monophosphates apparently non-competitively. However, aphidicolin does not inhibit the incorporation of dAMP into poly(dT) . oligo(A)10 nor does it inhibit the incorporation of dGMP into poly(dC) . oligo(dG)10, but, it does competitively inhibit the incorporation of dTMP into poly(dA) . oligo(dT)10.     

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).     

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.     

The adenovirus DNA binding protein and adenovirus DNA polymerase interact to catalyze elongation of primed DNA templates

The adenovirus-encoded 140-kDa DNA polymerase (Ad Pol) and the 59-kDa DNA binding protein (Ad DBP) are both required for the replication of viral DNA in vivo and in vitro. Previous studies demonstrated that, when poly(dT).oligo(dA) was used as a template-primer, both proteins were required for poly(dA) synthesis. In this report, the interaction between the Ad Pol and Ad DBP was further investigated using poly(dT).oligo(dA) as well as a linear duplex molecule containing 3' poly(dT) tails. DNA synthesis with the tailed template required Ad Pol, Ad DBP, and an oligo(dA) primer hydrogen bonded to the poly(dT) tails. Incorporation was stimulated 8-10-fold by ATP; however, no evidence of ATP hydrolysis to ADP was observed. Synthesis was initiated at either end of the tailed molecule and proceeded through the duplex region to the end of the molecule. This ability to translocate through duplex DNA and to synthesize long poly(dA) chains suggests that the Ad Pol.Ad DBP complex can act efficiently in the elongation reactions involved in the replication of Ad DNA (both type I and type II). During the replication reaction, substantial hydrolysis of deoxynucleoside triphosphates to the corresponding deoxynucleoside monophosphates occurred. This reaction required DNA synthesis and most likely reflects an idling reaction similar to that observed with other DNA polymerases containing 3'----5' exonuclease activity in which the polymerase first incorporates and then hydrolyzes a dNMP.     

Factor D is a selective single-stranded oligodeoxythymidine binding protein.

Factor D, a protein purified from rabbit liver that selectively enhances traversal of template oligodeoxythymidine tracts by diverse DNA polymerases, was examined for the sequence specificity of its binding to DNA. Terminally [32P]-labeled oligomers with the sequence 5'-d[AATTC(N)16G]-3', N being dT, dA, dG, or dC, were interacted with purified factor D and examined for the formation of protein-DNA complexes that exhibit retarded electrophoretic mobility under nondenaturing conditions. Whereas significant binding of factor D to 5'-d[AATTC(T)16G]-3' is detected, there is no discernable association between this protein and oligomers that contain 16 contiguous moieties of dG, dA, or dC. Furthermore, factor D does not form detectable complexes with the duplexes oligo(dA).oligo(dT) or poly(dA).poly(dT). The preferential interaction of factor D with single-stranded poly(dT) is confirmed by experiments in which the polymerase-enhancing activity of this protein is protected by poly(dT) against heat inactivation two- and four-fold more efficiently than by poly(dA) or poly(dA).poly(dT), respectively.     

Human cytomegalovirus. III. Virus-induced DNA polymerase.

Infection of WI-38 human fibroblasts with human cytomegalovirus (CMV) led to the stimulation of host cell DNA polymerase synthesis and induction of a novel virus-specific DNA polymerase. This cytomegalovirus-induced DNA polymerase was purified and separated from host cell enzymes by DEAE-cellulose and phosphocellulose column chromatographies. It can be distinguished from host cell enzymes by chromatographic behavior, template primer specificity, sedimentation property, and the requirement of salt for maximal activity. This virus-induced enzyme has a sedimentation coefficient of 9.2S and is found in both the nuclei and cytoplasm of virus-infected cells, but not in uninfected cells. This enzyme could efficiently use activated calf-thymus DNA, oly(dA)-oligo(dT)12-18, and poly(dC)-oligo(dG)12-18 as template primers, especially poly(dA)-oligo(dT)12-18, but it could not use poly(rA)-oligo(dT)12-18, poly(rC)-oligo(dG)12-18, or oligo(dT)12-18. The enzyme requires Mg2+ for maximal activity, is sensitive to p-hydroxymercuribenzoate, and is not a zinc metalloenzyme. In addition, the cytomegalovirus-induced DNA polymerase activity can be enhanced by adding 0.06 to 0.12 M NaCl or 0.03 to 0.06 M (NH4)2SO4 to the reaction mixture.     

Purification and characterization of porcine liver DNA polymerase gamma: utilization of dUTP and dTTP during in vitro DNA synthesis.

Porcine liver DNA polymerase gamma has been demonstrated to preferentially incorporate dTMP over dUMP during in vitro DNA synthesis. When polymerase activity was measured in standard reactions containing saturating levels of either dTTP or dUTP, the polymerization rate was slightly faster in the reaction containing dTTP. However, under conditions where both dTTP and dUTP competed, at an equal molar concentration, approximately 3-times more thymine residues were incorporated than uracil residues into DNA. Similarly, preferential incorporation of dTMP was observed on several substrates including poly (dA).oligo p(dT), poly (rA).oligo p(dT) and poly (dA-dT). The discrimination against dUMP incorporation was even more apparent with reduced levels of dUTP. These observations were consistent with the finding that the Km for DNA polymerase gamma was about 3-fold lower for dTTP (0.4 microM) than for dUTP (1.1 microM). On the other hand, the Vmax for these two reactions was very similar. Discrimination against dUMP incorporation was also observed during inhibition of polymerase gamma by dideoxyribonucleoside triphosphates. Dideoxythymidine triphosphate preferentially inhibited dUMP incorporation compared to that of dTMP, whereas ddATP, ddCTP and ddGTP inhibited both reactions equally.