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

Abstract

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.

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

“…At 45°C both the length of the products formed (12) and the intensity of the bands decreased (lane 5). As for processivity, DNA polymerase 2 activity shows a similar response to temperature (Coello et al, 1992).…”

“…At pH 5.9 a distributive behavior was observed. Optimal activity for maize DNA polymerase 2 had been found previously to be at pH 7.0 (Coello et al, 1992). The optimal KC1 concentration for DNA polymerase 2 activity is 100 mM, and its activity is progressively inhibited by higher KC1 concentrations (Coello et al, 1992).…”

“…Enzymes with similar properties to DNA polymerase a have been studied in periwinkle (Gardner and Kado, 1976), sugar beet (Tymonko and Dunham, 1977), rice (Amileni et al, 1979), spinach (Misumi and Weissbach, 1982), pea (Bryant et al, 1992), wheat (Laquel et al, 1990;Balmukhanov et al, 1992), and maize (Zen mnys L.) (Coello et al, 1992). In maize, this enzyme, designated DNA polymerase 2, requires Mg2' for activity and is stimulated by K+.…”

“…It has K , values for the different dNTPs that are similar to those reported for animal enzymes, and has an optimal pH of 7.0 and an optimum temperature of 37°C. The enzyme cross-reacts with an antibody developed against calf thymus DNA polymerase a (Coello et al, 1992). Western blot analysis, using an antibody raised against the maize enzyme, indicates the presence of 90-, 83-, 70-, 60-, 55-, 45-, and 24-kD subunits (Coello et al, 1994), with the 90-kD subunit possessing the catalytic activity.…”

Regulating DNA Replication in Plants

“…The presence of DNA polymerase a (POLA) activity in plants has been shown biochemically in maize, wheat, pea, and cauliflower (Bryant 1980;Castroviejo et al 1990;Laquel et al 1990;Balmukhanovet al 1992;Bryant et al 1992;Coello et al 1992;Coello and Vazquez-Ramos 1995b;Garcia et al 1997;Luque et al 1998). More recently, the genome sequence analysis of Arabidopsis and rice allowed the identification of the four putative subunits of POLA complex (POLA1 to 4).…”