Comparison among DNA polymerases 1, 2 and 3 from maize embryo axes. A DNA primase activity copurifies with DNA polymerase 2.

Three DNA polymerase activities, named 1, 2 and 3 were purified from maize embryo axes and were compared in terms of ion requirements, optimal pH, temperature and KCl for activity, response to specific inhibitors and use of templates. All three enzymes require a divalent cation for activity, but main differences were observed in sensitivity to inhibitors and template usage: while DNA polymerases 1 and 2 were inhibited by N-ethyl maleimide and aphidicolin, inhibitors of replicative-type enzymes, DNA polymerase 3 was only marginally or not affected at all. In contrast, DNA polymerase 3 was highly inhibited by very low concentrations of ddTTP, an inhibitor of repair-type enzymes, and a 100-fold higher concentration of the drug was needed to inhibit DNA polymerases 1 and 2. Additionally, DNA polymerases 1 and 2 used equally or more efficiently the synthetic template polydA-oligodT, as compared to activated DNA, while polymerase 3 used it very poorly. Whereas DNA polymerases 1 and 2 shared properties of replicative-type enzymes, DNA polymerase 3 could be a repair-type enzyme. Moreover, a DNA primase activity copurified with the 8000-fold purified DNA polymerase 2, strengthening the suggestion that polymerase 2 is a replicative enzyme, of the alpha-type. This DNA primase activity was also partially characterized. The results are discussed in terms of relevant data about other plant DNA polymerases and primases reported in the literature.

Studies on the Processivity of Maize DNA Polymerase 2, an [alpha]-Type Enzyme.

This paper describes studies on the processivity of an [alpha]-type DNA polymerase from maize (Zea mays L.) embryonic axes, designated as DNA polymerase 2. Using poly(dA)/oligo(dT) as template, DNA polymerase 2 has a processivity of 18 ([plus or minus]5) nucleotides incorporated, a value much lower than that found for wheat [alpha]-type DNA polymerase (P. Laquel, S. Litvak, M. Castroviejo [1993] Plant Physiol 102: 107-114). Conditions that maximally stimulate enzyme activity, such as 100 mM KCl and 12 mM Mg2+, are strongly inhibitory of processivity and cause the enzyme to become distributive under these conditions. Optimal concentrations for processivity are 10 mM KCl and 1 to 2 mM Mg2+. Both enzyme activity and processivity were found to be similar at different Mn2+ concentrations. Both DNA polymerase 2 activity and processivity are greatly reduced by spermine and N-ethylmaleimide. A distinguishing feature of processivity in DNA polymerase 2 was the response to ATP, which not only stimulated processivity by more than 2-fold, but also produced a distinctive pattern in which the enzyme seemed to pause every 10 nucleotides, reaching a value of 40 to 50 nucleotides incorporated. This pattern was observed in some, but not all, heparin-Sepharose fractions with enzyme activity, suggesting the possibility of different DNA polymerase 2 complexes.

Maize DNA polymerase 2 is a phosphoprotein with increasing activity during germination.

DNA replication is a late event during maize germination and DNA polymerase activity increases as germination proceeds. A replicative alpha-type DNA polymerase has been purified from maize seeds (DNA polymerase 2) and has been shown to be a multisubunit complex [Coello, P., Rodríguez, R., García, E. & Vázquez-Ramos, J. M. (1992) Plant Mol. Biol. 20, 1159-1168; Coello, P., García, E. & Vázquez-Ramos, J. M. (1994) Can. J. Botany 72, 818-822]. DNA polymerase 2 activity increased several fold during maize germination, with no apparent change in either the amount of holoenzyme or in any of the individual subunits. However, the level of phosphorylation of the 90-, 70-, 55- and 45-kDa polypeptides changed in a cyclic fashion with their highest levels occurring at 11-14 h and 45-48 h of germination. Phosphate incorporated into the different polypeptides in the 11-14-h period remained stable for at least the next 10 h (to 24 h of germination), the period of maximal enzyme activity. However, DNA polymerase 2 processivity was very similar in freshly prepared 3-h and 24-h enzymes, and no evidence was found that polymerase activity was modified by in vitro phosphorylation. The significance of these results is discussed.

A DNA polymerase from maize axes: its purification and possible role.

Three different DNA polymerase activities can be resolved by passing a protein extract from 24 h imbibed maize axes through DEAE-cellulose. These activities have been numbered 1, 2 and 3, according to their elution order. One of them, DNA polymerase 2, elutes at 100-120 mM phosphates. This enzyme was further purified by passing it through Heparin-Sepharose, Sephacryl S-300 and DNA cellulose. Purification was nearly 5000-fold. The enzyme needs Mg2+, is stimulated by K+, has an optimum pH of 7.0 and its optimum temperature is 30-37 degrees C. Specific inhibitors for different types of polymerases, such as aphidicolin, dideoxythymidine triphosphate and N-ethyl maleimide, gave intermediate values of inhibition, making impossible the definition of the type of enzyme purified by its inhibitory pattern. SDS-PAGE indicated the presence of several bands of molecular masses of 28-40, 56 and 15 kDa. Most of these bands could be visualized when proteins from crude extracts were analyzed by western blot, using an antibody against calf thymus DNA polymerase alpha. A high molecular mass (around 500 kDa) was calculated by western blot of native gels using the same antibody. Finally, specific activity of this enzyme increased 100-fold during maize germination whereas polymerase 3 virtually did not increase. Furthermore, immunoprecipitation experiments with the antipolymerase alpha-antibody showed a decrease in DNA polymerase activity by 70%. The possibility that polymerase 2 is a replicative enzyme is discussed.

Oxolinic acid-resistant mutants of Bacillus subtilis.

Two mutants of Bacillus subtilis have been independently selected for resistance to oxolinic acid, and inhibitor of DNA gyrase. The mutations, designated oxr-1 and oxr-2, map very close to one another but are clearly separated from mutations in the genes for DNA gyrase. Many of the phenotypic properties of the mutants differ from those of a strain containing the gyrA mutation described by other workers. In particular, the oxr strains are as sensitive as the wild-type to inhibition by nalidixic acid on solid medium. In addition, experiments with DNA synthesis in toluenized cells show that the enzyme of the gyrA mutant is resistant to oxolinic acid, whereas DNA synthesis in the oxr mutants is as sensitive as it is in wild-type preparations. It is concluded that resistance to oxolinic acid is not due to an alteration in the DNA gyrase, but is more probably the result of an impaired uptake of the inhibitor. Although growth of the mutants on agar plates is inhibited at high concentrations of oxolinic acid, lower concentrations (1-2 microgram ml-1) can be used to distinguish them from the wild-type. The oxr-1 and oxr-2 mutations define a new genetic locus and can be used as genetic markers in B. subtilis.

Inhibition of sporulation by DNA gyrase inhibitors.

The effects of oxolinic acid and novobiocin - which respectively inhibit the A and B subunits of DNA gyrase, and therefore inhibit DNA synthesis - have examined in sporulating cultures of Bacillus subtilis. Although both inhibitors prevent sporulation, this is not due to inhibition of DNA synthesis. Instead, they affect protein synthesis generally, though weakly, but have very marked effects on the formation of individual enzymes. These effects are reproducible, but the type of enzyme that will be affected is not predictable. The results point to an involvement of DNA gyrase in the transcription of some genes. This is suggested as the reason for the effect of the inhibitors on spore formation, which they block mainly at Stage O-I.