Molecular cloning of the cDNA for the catalytic subunit of plant DNA polymerase alpha and its cell-cycle dependent expression.

Abstract:

BACKGROUND: DNA polymerase alpha has been studied in considerable detail in yeast and animals. Genetic and biochemical analyses reveal that this enzyme is composed of a heterotetramer and is necessary for replicon initiation and primer synthesis in lagging strand synthesis. In spite of the fact that modes of DNA replication in plants seem to be similar to those in other eukaryotes, very little is known about the biochemical components that participate in DNA replication of plants, including DNA polymerases. RESULTS: Using a 561-base pair DNA fragment, obtained by polymerase chain reaction amplification from a rice cDNA library as a probe, we isolated and sequenced a cDNA homologous to the cDNA for the catalytic subunit of rice DNA polymerase alpha. The encoded polypeptide has extensive homology with the catalytic subunit of DNA polymerase alpha from several species. Furthermore, when the cDNA was expressed in eukaryotic transcription/translation systems, the protein products showed DNA polymerase activity which was inhibited by a monoclonal antibody specific for DNA polymerase alpha. Using RNA gel blot analysis, we found that the levels of mRNA of the catalytic subunit of this enzyme is regulated during the cell-cycle in plant cells. CONCLUSION: This is the first report which describes the cDNA cloning of plant DNA polymerase. We conclude that the principal features of the DNA polymerase alpha catalytic subunit are conserved in plants.

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