tailieunhanh - Báo cáo khoa học: Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae

DNA polymerasec, a mitochondrial replication enzyme of yeasts and ani-mals, is not present in photosynthetic eukaryotes. Recently, DNA polyme-rases with distant homology to bacterial DNA polymerase I were reported in rice, Arabidopsis, and tobacco, and they were localized to both plastids and mitochondria. | ỊFEBS Journal Purification and characterization of organellar DNA polymerases in the red alga Cyanidioschyzon merolae Takashi Moriyama Kimihiro Terasawa Makoto Fujiwara and Naoki Sato Department of Life Sciences Graduate Schoolof Arts and Sciences University of Tokyo Japan Keywords Cyanidioschyzon merolae DNA polymerase organelle POP replication Correspondence N. Sato Department of Life Sciences Graduate School of Arts and Sciences University of Tokyo Komaba Meguro-ku Tokyo 153-8902 Japan Fax 81 3 5454 6698 Tel 81 3 5454 6631 E-mail naokisat@ Received 28 January 2008 revised 30 March 2008 accepted 1 April 2008 doi DNA polymerase c a mitochondrial replication enzyme of yeasts and animals is not present in photosynthetic eukaryotes. Recently DNA polymerases with distant homology to bacterial DNA polymerase I were reported in rice Arabidopsis and tobacco and they were localized to both plastids and mitochondria. We call them plant organellar DNA polymerases POPs . However POPs have never been purified in the native form from plant tissues. The unicellular thermotrophic red alga Cyanidioschyzon mero-lae contains two genes encoding proteins related to Escherichia coli DNA polymerase I PolA and PolB . Phylogenetic analysis revealed that PolB is an ortholog of POPs. Nonphotosynthetic eukaryotes also have POPs which suggested that POPs have an ancient origin before eukaryotic photosynthesis. PolA is a homolog of bacterial DNA polymerase I and is distinct from POPs. PolB was purified from the C. merolae cells by a series of column chromatography steps. Recombinant protein of PolA was also purified. Sensitivity to inhibitors of DNA synthesis was different in PolA PolB and E. coli DNA polymerase I. Immunoblot analysis and targeting studies with green fluorescent protein fusion proteins demonstrated that PolA was localized in the plastids whereas PolB was present in both plastids and mitochondria. The expression of PolB was .