Biochemical characterization of DNA polymerase delta and cloning of its cDNA from the Glycine max cell line, SB-M

Jeannie T. B. Collins

Abstract

Characterization of the plant $\alpha$-like and $\delta$-like nuclear DNA polymerases from the soybean (Glycine max) cell line SB-M was performed. Antibodies directed against mammalian DNA polymerases $\alpha$ and $\delta$ crossreacted with partially purified DNA polymerases from the SB-M cells. An antibody against mammalian accessory protein for DNA polymerase $\delta$, Proliferating Cell Nuclear Antigen (PCNA), also recognized a protein from the plant cells. Inhibitor studies supported the classification of one of the plant DNA polymerases as $\alpha$-like. The plant $\alpha$-like DNA polymerase was sensitive to butylphenyl dGTP and aphidicolin, was moderately inhibited by arabinosyl CTP (araCTP), and was sensitive to the sulfhydryl reagent N-ethylmaleimide (NEM). A second DNA polymerase activity was tentatively labeled as $\delta$-like, however, its response to DNA polymerase inhibitors indicated the fraction with this activity may also contain $\varepsilon$- or $\beta$-like DNA polymerase activities. The DNA polymerase in this fraction was sensitive to NEM, moderately inhibited by araCTP and was insensitive to aphidicolin and butylphenyl dGTP. The protein in this fraction was recognized by antibodies against calf thymus Pol $\delta$, supporting the presence of a $\delta$-like DNA polymerase, while protein in the fraction containing the $\alpha$-like DNA polymerase did not crossreact. These biochemical similarities and antibody crossreactivity suggested that there may be conserved regions among plant, animal and yeast DNA polymerases. Degenerate PCR primers that were based on conserved amino acid regions common to animal and yeast DNA polymerases were employed to amplify a 1300 base pair product from SB-M cDNA. Using the nucleotide sequence of this product, the 3$\sp\prime$ and 5$\sp\prime$ ends of the cDNA were amplified by Rapid Amplification of cDNA Ends (RACE). The composite cDNA was 3476 nucleotides long, encompassing an open reading frame of 3267 bases that can encode a protein of 1088 amino acids. The derived amino acid sequence showed considerable homology to DNA polymerase $\delta$ from Candida, Drosophila, mouse and human, especially within the region that contains the conserved amino acid sequences in the central portion of the deduced sequence.