A low molecular weight DNA polymerase from wheat embryos

Abstract

The study of plant DNA polymerases lags far behind that concerning their animal or yeast counterpart. In this work we describe the first extensive purification to apparent homogeneity, as well as a detailed biochemical and immunological characterization, of a low molecular weight DNA polymerase (DNA polymerase CI) purified from wheat embryos. The monomeric enzyme is a basic protein having a molecular weight of 52 kDa. Polyclonal antibodies raised in rabbits against DNA polymerase CI did not inhibit animal DNA polymerases alpha and beta or wheat DNA polymerase A, whereas wheat DNA polymerases CII and B were much less affected than the CI enzyme. Several properties of enzyme CI were studied. Some known inhibitors of DNA polymerase activity including aphidicolin, phosphonoacetic acid and heparin, did not affect DNA polymerase CI while the activity of this enzyme was strongly inhibited by ddTTP and N-ethylmaleimide. The polyamine spermine decreased markedly the enzyme activity, while spermidine produced a strong stimulation at the same concentrations that spermine inhibited the enzyme. The best template for this enzyme is poly dA-oligo dT, although polymerase CI can recognize significantly some synthetic polyribonucleotide templates (poly rC-oligo dG, poly rA-oligo dT) but only at a given protein/template primer ratio. The enzyme is blocked at the amino terminus, thus preventing the automatic sequencing of the protein. The amino acid analysis showed a striking similarity with the animal low molecular weight DNA polymerase beta. The latter observation, as well as the effect of inhibitors (except N-ethylmaleimide which does not inhibit the animal polymerase) indicate that the DNA polymerase described in this work is a plant DNA polymerase very similar to the low molecular weight animal DNA polymerase beta, an enzyme believed to be involved in nuclear DNA repair.

Changes of enzymes and factors involved in DNA synthesis during wheat embryo germination

“…Either wheat germ or wheat embryos (4-8 g) were used. Ammonium sulfate fractionation, and purification by phosphocellulose, DEAE cellulose and MonoS chromatography were performed as described before [6].…”

“…In our laboratory, four wheat germ DNA polymerases from nuclear origin, named A, B, CI and CII, have been purified and characterized [4,6,29]. A comparative analysis of their biochemical properties with mammalian DNA polymerases does not show a strong similarity between plant and animal systems.…”

Wheat DNA Primase (RNA Primer Synthesis in Vitro, Structural Studies by Photochemical Cross-Linking, and Modulation of Primase Activity by DNA Polymerases)

“…The detailed purification procedures of DNA primase (Laque1 et al, 1990a) and DNA polymerases A, B, (31, and CII have been published previously (Castroviejo et al, 1979(Castroviejo et al, , 1991Lalquel et al, 1990b;Richard et al, 1991).…”

“…We found that DNA polymerase CII has some properties of DNA polymerase a, whereas DNA polymerase B is very similar to DNA polymerase 6, since it is highly resistant to butyl-phenyl dGTP and possesses a 3'-5' exonuclease activity (Richard et al, 1991). DNA polymerase CI is similar to animal DNA polymerase p: it is the only low mo1 wt DNA polymerase in wheat and it is resistant to aphidicolin and highly sensitive to ddTTP (Castroviejo et al, 1991). DNA polymerase A is an difficult enzyme to classify and shows some properties of DNA polymerase y (Tarrago-Litvak et al, 1975).…”

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).…”