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A procedure has been developed to assess whether polymerization of nucleotides by DNA polymerases is processive, that is, whether a succession of polymerization steps occurs without release of the enzyme from the template. The method involves measurement of the ratio of deoxyguanylate to deocycytidylate incorporated in the course of replicating a segment of the right hand cohesive end of phage lambaDNA with the sequence 5'-G-G-G-C-G-G-C-G-3'. In the case of the Escherichia coli DNA polymerase I, each enzyme molecule completes synthesis of the sequence before dissociation occurs. Furthermore, at both 6 and 22 degrees, the polymerase remains bound to the lambdaDNA template after synthesis has completed. Template challenge experiments, in which the polymerase is allowed to begin synthesis in the presence of a molar excess of lambdaDNA before addition of a very large excess of calf thymus DNA, show that under the conditions used, productive binding of polymerase to lambdaDNA is a slow process requiring 1 to 2 hours. After synthesis has been completed, polymerase remains bound to the lambdaDNA in spite of the availability of new primer termini. The association, polymerization, and dissociation rates measured in these experiments suggest that the polymerization reaction catalyzed by DNA polymerase I is processive, and that hundreds of nucleotides may be polymerized between each association and dissociation.

The T4 DNA polymerase catalyzes extensive exonucleolytic degradation of linear duplex DNA starting at the 3' ends. However, the enzyme will release only a few nucleotides from each DNA molecule if the degradation is run in the presence of a single deoxynucleoside triphosphate. Several experiments suggested that the polymerase degrades a chain only until a nucleotide is released which can be replaced by transfer from the triphosphate. Subsequent reaction consists only of alternating removal and replacement of that nucleotide, without net degradation of the DNA. Because only a few nucleotides are released in the presence of a single triphosphate, it seemed possible that the polymerase could be used for analysis of nucleotide sequences at 3' termini of duplex DNA. This possibility was verified by the analysis of the 3'-terminal sequence of fragments of bacteriophage T7 DNA produced by endonuclease R, the Hemophilus influenzae restriction endonuclease. This terminal sequence had been previously identified by Kelly and Smith (J. Mol. Biol., 51, 393 (1970)). The method used in this analysis, which should also be applicable to other DNAs, was to degrade 32P-labeled DNA by the T4 polymerase in the presence of each of the four triphosphates. The nucleotides released in each case were identified and quantitatively determined by thin layer chromatography, and were consistent with the previously determined terminal sequence.

We have purified the small nuclear DNA polymerase from cultured KB cells to ≥95% homogeneity. Electrophoresis on sodium dodecyl sulfate polyacrylamide gel slabs demonstrates that the polymerase is comprised of a single polypeptide chain of molecular weight 43,000, and this size is in agreement with that of the polymerase activity as determined by gel filtration. Although the enzyme carries out a conventional polymerization reaction, incorporating complementary deoxynucleoside triphosphates at 3'-hydroxyl termini with concomitant stoichiometric release of PPi, it cannot be demonstrated to carry out PPi exchange. The polymerase is devoid of exonuclease activities, particularly the 3'→5'-exonuclease activity, as tested by the most sensitive available assays. However, the enzyme does exhibit prominent primer-template-dependent conversion of deoxynucleoside triphosphate to deoxynucleoside monophosphate. We propose that this reaction represents an independent polymerase-associated activity that is unrelated to 3'→5'-exonuclease, in contrast to the model that has been developed from studies with prokaryotic polymerases. Finally, the KB polymerase is unable to excise mismatched primer termini from a synthetic homopolymer primer-template, but rather can utilize such termini as functional initiation sites for polymerization.

A simple and rapid method for determining nucleotide sequences in single-stranded DNA by primed synthesis with DNA polymerase is described. It depends on the use of Escherichia coli DNA polymerase I and DNA polymerase from bacteriophage T4 under conditions of different limiting nucleoside triphosphates and concurrent fractionation of the products according to size by ionophoresis on acrylamide gels. The method was used to determine two sequences in bacteriophage φX174 DNA using the synthetic decanucleotide A-G-A-A-A-T-A-A-A-A and a restriction enzyme digestion product as primers.

Publisher Summary This chapter describes the assay method, isolation procedure, and properties of T4 DNA polymerase. The hydroxyapatite fraction of the T4 DNA polymerase is a homogeneous protein. The T4 DNA polymerase consists of a single polypeptide chain with a molecular weight of 114,000. T4 DNA polymerase requires a single-stranded template with a free 3'-hydroxyl terminus that is replicated to an extent that approaches but never exceeds the DNA input. The product is a helical structure in which the newly synthesized strand is covalently linked from its 5' terminus to the 3' terminus of the template. T4 DNA polymerase activity is markedly and specifically stimulated by the T4 gene 32 protein. The chapter discusses the interaction of polymerase and 3'→5' exonuclease activities. It also discusses the mutants of T4 DNA polymerase.

Summary E. coli DNA polymerase I has been used to synthesize DNA complementary to rabbit globin mRNA. In addition to the heteropolymeric DNA, poly (dT) and poly (dA)·(dT) are also synthesized. The extent of synthesis of these three products decreases at different rates upon heat inactivation of the polymerase, suggesting that the sites of synthesis on the enzyme are either entirely or partially separate. The use of polymerase I for copying RNAs makes possible the ready availability of complementary DNAs.

Much interest of late has centered around RNAdirected DNA synthesis. Initially, with the finding of an RNA-dependent DNA polymerase in oncogenic RNA viruses [l-S] and what was described as the exclusive presence of the polymerase in acute leukemia cells as opposed to ‘normal’ cells [6] , speculation as to a molecular basis for leukemogenesis and a predictive test for leukemia abounded. However, with the identification [7, S] of the polymerase in ‘normal’ cells (cells not known to be RNA virally transformed) the hope of an absolute predictive index for leukemia or neoplasia has lost credibility, and the RNA-dependent DNA polymerase may be of more interest from the standpoint of gene amplification as opposed to a neoplastic predictor. In our studies of this polymerase we have undertaken an extensive, systematic experiment designed to note the effects of a number of potential inhibitors (primarily antibiotics) on the RNAdependent DNA polymerase in vitro. The present brief report gives the results of these experiments. Several compounds were shown to be potent inhibitors of an RNA-dependent DNA from mouse leukemia cells.

The dependence of in vitro RNA synthesis, catalyzed by purified Escherichia coli RNA polymerase, on the concentration and secondary structure of T4 DNA templates in the reaction mixture has been studied by means of kinetic and sedimentation experiments. The following results were obtained. (1) If the weight ratio of DNA to polymerase in the reaction mixture is low, then the rate of RNA synthesis is proportional to the DNA concentration and nearly independent of the polymerase concentration (saturation of DNA with polymerase). If that ratio is high, then the rate of RNA synthesis is proportional to the polymerase concentration and nearly independent of the DNA concentration (saturation of polymerase with DNA). For intermediate ratios, the ensuing rate of RNA synthesis in the reaction mixture can be increased by addition of either more polymerase or more DNA, suggesting that both polymerase and starting sites for polymerase on the DNA are heterogeneous with respect to their mutual binding affinities. (2) The rate of chain growth of the RNA is independent of the DNA concentration; accordingly, at limiting DNA concentration and fixed polymerase concentration, only the number of polymerase molecules becoming active is increased with increasing DNA concentration. (3) The maximum number of long RNA molecules that can be synthesized simultaneously, and thus the maximum number of polymerase molecules that can be fully active per native T4 DNA unit, is estimated to be 180. (4) On heat-denatured T4 DNA, the maximum number of RNA molecules that can be synthesized per unit weight of DNA is several times higher than on native T4 DNA. To account for these results, a model is proposed which assumes a limited number of specific starting sites for RNA polymerase on the native T4 DNA molecule.